Wir möchten uns für die Unterstützung bei Patienten Gewebeproben Sammlung danke den Mitarbeitern des Tissue Beschaffungsmöglichkeiten der Universität von Minnesota. Diese Arbeit wurde durch das Department of Defense Eierstockkrebs-Forschungsprogramm (OCRP) OC093424 zu MB, die von der Randy Shaver Krebsforschung und Gemeinschaftsfonds zur MB, von der Minnesota-Eierstockkrebs-Allianz zu MB und durch die Gynäkologische Onkologie Abteilungs Fonds MB. Die Geldgeber hatten keine Rolle in Studiendesign, Datenerfassung und Analyse, Entscheidung zur Veröffentlichung oder der Zubereitung des Manuskripts.

Ethikerklärung Feste Proben von Eierstockkrebs wurden von der Universität von Minnesota Tissue Beschaffung Facility (TPF) nach Institutional Review Board Committee erhalten: Human Betreff Board (IRB)-Zulassung. 1. Reagenz-Setup <ol><li> Vorbereitung komplettem DMEM Medium durch Hinzu DMEM mit 10% FBS und 100 Einheiten Penicillin-Streptomycin. Speichert das Medium bei 4 ° C und warm auf 37 ° C vor der Verwendung. </li><li> Aliquot Dispase II in separate Bände 5-10 ml mehreren Gefrier vermeiden taut und bei -20 ° C in einem nonfrost freien Gefrierschrank. </li></ol> 2. Gewebeentnahme <ol><li> Sammeln feste Proben von Eierstockkrebs (~ 5 g in Größe) zum Zeitpunkt der Operation makroskopisch aus Bereichen wie Krebs von einem Pathologen identifiziert. Klinische Proben werden anonymisiert und nach institutionellen Protokolle eingetragen. </li><li> Zeigen Proben in einem sterilen, screw Deckel, Polypropylen Proben Behälter mit 30 ml eiskaltem PBS gefüllt. Transport der Proben von der chirurgischen Pathologie Labor in die Forschungslabor für die Verarbeitung auf Eis, und innerhalb von 30 Minuten von der Probenentnahme (Abbildung 1). </li></ol> 3. Gewebeverarbeitung <ol><li> Arbeiten unter sterilen Bedingungen, übertragen Sie die Proben auf einer Petrischale (60 mm x 60 mm) mit 10 ml frischem, eiskaltem PBS und mit einer sterilen Rasierklinge, weiter in die kleinstmögliche (2 mm oder weniger) Stücke geschnitten ( Figuren 2A und 2B). </li><li> Das zerkleinerte Gewebe übertragen in einen 15 ml konischen Röhrchen mit 10 ml vorgewärmtem (37 ° C für 30 min) Dispase II (2,4 U / ml) in DMEM und Inkubation bei 5% CO 2 und 37 ° C für 30 min. Um eine optimale Verdauung der Proben zu gewährleisten, schüttelt die Hand Zellaufschlämmung alle 5 min. </li><li> Nach 30 min Inkubation Übertragung (mit10 ml serologische Pipette) die Zelle Aufschlämmung auf eine Zelle Sieb (70 um Maschenweite) auf einem konischen 50 ml-Röhrchen gegeben und Anwendung eines leichten Druck gegen das Gitter unter Verwendung eines Spritzenkolbens. Verwerfen undissoziierter Gewebe (noch auf der Oberseite des Mesh) und sammeln Sie die erhaltene Zellsuspension in der 50 ml konischen Röhrchen steril. Zentrifuge bei 320 × g für 7 min bei 4 ° C (Fig. 3). </li><li> Verwerfen des Überstandes und Resuspendieren des Zellpellets in 10 ml DMEM mit 10% FBS. </li><li> Inkubieren der Zellsuspension in einer Petrischale mit 5% CO 2 und 37 ° C (Fig. 4). </li><li> Ändern Sie das Medium nach 24 Stunden von der ersten Beschichtung. Dies ermöglicht die Entfernung der Zelltrümmer und die Mehrzahl der in der Kultur anwesend Erythrozyten. </li><li> Ändern Sie das Medium alle drei Tage für die folgenden zwei Wochen, nach der die Kulturen von primären EOC sind bereit für Downstream-Anwendungen. </li></ol>

<ol>
	<li>Kurman, R. J., Visvanathan, K., Roden, R., Wu, T. C., Ie M, S. h. i. h. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+and+treatment+of+ovarian+cancer:+shifting+from+early+stage+to+minimal+volume+of+disease+based+on+a+new+model+of+carcinogenesis.">Early detection and treatment of ovarian cancer: shifting from early stage to minimal volume of disease based on a new model of carcinogenesis.</a> Am. J. Obstet. Gynecol. 198 (4), 351-356 (2008).</li><li>Kuhn, E., Kurman, R. J., Shih, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+Cancer+Is+an+Imported+Disease:+Fact+or+Fiction.">Ovarian Cancer Is an Imported Disease: Fact or Fiction.</a> Curr. Obstet. Gynecol. Rep. 1 (1), 1-9 (2012).</li><li>Sarojini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+biomarkers+for+ovarian.">Early detection biomarkers for ovarian.</a> J. Oncol. , (2012).</li><li>Shepherd, T. G., Theriault, B. L., Campbell, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nachtigal+M.W.+Primary+culture+of+ovarian+surface+epithelial+cells+and+ascites-derived+ovarian+cancer+cells+from+patients.">Nachtigal M.W. Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients.</a> Nat. Protoc. 1 (6), 2643-2649 (2006).</li><li>Tsao, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+ovarian+surface+epithelial+cells+immortalized+by+human+papilloma+viral+oncogenes+(HPV-E6E7+ORFs).">Characterization of human ovarian surface epithelial cells immortalized by human papilloma viral oncogenes (HPV-E6E7 ORFs).</a> Exp. Cell. Res. 218 (2), 499-507 (1995).</li><li>Auersperg, N., Maines-Bandiera, S. L., Dyck, H. G., Kruk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cultured+human+ovarian+surface+epithelial+cells:+phenotypic+plasticity+and+premalignant+changes.">Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes.</a> Lab. Invest. 71 (4), 510-518 (1994).</li><li>Brewer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+model+of+normal,+immortalized+ovarian+surface+epithelial+and+ovarian+cancer+cells+for+chemoprevention+of+ovarian+cancer.">In vitro model of normal, immortalized ovarian surface epithelial and ovarian cancer cells for chemoprevention of ovarian cancer.</a> Gynecol. Oncol. 98 (2), 182-192 (2005).</li><li>Sueblinvong, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment,+characterization+and+downstream+application+of+primary+ovarian+cancer+cells+derived+from+solid+tumors.">Establishment, characterization and downstream application of primary ovarian cancer cells derived from solid tumors.</a> PLoS One. 7 (11), (2012).</li><li>Rockwell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo-in+vitro+tumour+cell+lines:+characteristics+and+limitations+as+models+for+human+cancer.">In vivo-in vitro tumour cell lines: characteristics and limitations as models for human cancer.</a> Br. J. Cancer Suppl. 41 (4), 118-122 (1980).</li><li>Wong, C., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development,+application+and+limitations+of+breast+cancer+cell+lines+to+study+tamoxifen+and+aromatase+inhibitor+resistance.">The development, application and limitations of breast cancer cell lines to study tamoxifen and aromatase inhibitor resistance.</a> J. Steroid. Biochem. Mol. Biol. 131 (3-5), 83-92 (2012).</li></ol>

Die Autoren haben nichts zu offenbaren.

Ein besseres Verständnis der Ätiologie und Eierstockkrebs-Entwicklung ist entscheidend für die Verbesserung der Ergebnisse der Frauen, die von dieser schweren Krankheit betroffen. In diesem Zusammenhang ist der Einsatz von etablierten und &quot;handelsüblichen&quot; Eierstockkrebs-Zelllinie ist zweifellos enorm nützlich. Doch heute wissen wir, dass Krebszelllinien sind nicht repräsentativ für den menschlichen Tumor sie aus in viele Aspekte, einschließlich der Mangel an Heterogenität 9,10 entstanden. ...

Trotz seiner relativ geringen Inzidenz, Eierstockkrebs ist die tödlichste der gynäkologischen Krankheiten und die fünfthäufigste Ursache für Todesfälle durch Krebs bei Frauen 1,2. Dies ist vor allem auf den Mangel an zuverlässigen Tools und Modelle, die treu zu rekapitulieren die Initiierung und Progression der Erkrankung 3. Die meisten unserer heutigen Wissen über Eierstockkrebs wurde durch die Verwendung von Eierstock verewigt Oberfläche Epithelzellen (IOSen), Eierstockkrebs-Zelllinien und primären Eierstockkrebszellen aus Aszites 4-7 gewonnen möglich. Leider hat die Verwendung mehrere Einschränkungen, einschließlich einer Reihe von genetischen und phänotypischen Änderungen Immortalisierung Verfahren oder in vitro-Durchgänge zugeordnet ist, und die Heterogenität der Population von Aszitesflüssigkeit Herstellung resultieren. Daher primären Eierstockkrebszellen aus identifizierbaren und spezifischen festen Proben von Eierstockkrebs abgeleitet sind eine unique Werkzeug für die Untersuchung Eierstockkrebs Progression. Die Hauptschwierigkeiten in den Erhalt dieser bösartigen Zellen beteiligt sind auf ein übermäßiges Wachstum von Stromazellen oder Fibroblasten mit Verlust der Lebensfähigkeit und vorzeitige fehlende Proliferationskapazität in der Kultur dieser Zellen EOC. Verschiedene Methoden zur Einzelzellsuspensionen aus soliden Tumoren zu erstellen derzeit existiert, durch mechanische Mittel oder enzymatische Dissoziation jedoch bestimmte Techniken ergeben eine größere Menge des bevorzugten Ergebnis 8. Hier zeigen wir, dass die enzymatische Verdauung mit Dispase II führt zu einer effektiven Wiederherstellung lebensfähiger, Fibroblasten frei EOC-Zellen. Die so erhaltenen Kulturen EOC sind sehr anfällig für genetische Manipulation und sind in Arzneimittel-Screening-Tests, was anzeigt, dass diese EOC Kulturen sind für viele Anwendungen abwärts.

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			<td height="21" style="height:21px;">&#160;Small dissecting scissors, sterile</td>
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			<td height="40" style="height:40px;">&#160;15 ml conical capped tubes, sterile (BD Falcon)</td>
			<td style="width:125px;">BD, Falcon</td>
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			<td height="40" style="height:40px;">50 ml conical capped tubes, sterile (BD Falcon)</td>
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			<td align="right" style="width:155px;">352027</td>
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			<td height="21" style="height:21px;">&#160;10 ml serological pipette, sterile&#160;</td>
			<td style="width:125px;">Corning</td>
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			<td height="21" style="height:21px;">6 ml syringe plunger, sterile&#160;</td>
			<td style="width:125px;">Tyco Healthcare</td>
			<td align="right" style="width:155px;">8881516911</td>
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			<td height="40" style="height:40px;">Nylon filter (70 &#956;m ) cell strainer, sterile (BD Falcon)</td>
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			<td align="right" style="width:155px;">352350</td>
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			<td height="21" style="height:21px;">5-cm Petri dish, sterile&#160;</td>
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			<td height="21" style="height:21px;"></td>
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			<td height="21" style="height:21px;">10-cm Petri dish, sterile&#160;</td>
			<td style="width:125px;">Corning</td>
			<td align="right" style="width:155px;">430167</td>
			<td></td>
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method for obtaining primary ovarian cancer cells from solid specimens

Zuverlässige Werkzeuge für die Untersuchung von Eierstockkrebs Initiierung und Progression sind dringend erforderlich. Während die Verwendung von Eierstock-Krebszelllinien bleibt ein wertvolles Werkzeug für das Verständnis von Eierstock-Krebs hat ihr Einsatz viele Einschränkungen. Dazu gehören der Mangel an Heterogenität und die Fülle von genetischen Veränderungen mit erweiterten In-vitro-Passage verbunden. Hier beschreiben wir eine Methode, die für eine schnelle Einrichtung der primären Eierstockkrebszellen bilden können feste klinischen Proben zum Zeitpunkt der Operation gesammelt. Das Verfahren besteht aus der klinischen Proben zur enzymatischen Verdau für 30 min. Die isolierten Zellsuspension wird erlaubt, zu wachsen und kann für Folgeanwendungen einschließlich Arzneimittel-Screening verwendet werden. Der Vorteil der primären Eierstockkrebszelllinien gegenüber etablierten Eierstock-Krebszelllinien ist, dass sie repräsentativ für die ursprünglichen spezifischen klinischen Proben sie von abgeleitet und kann von verschiedenen Stellen abgeleitet werden, ob es primary oder metastasierenden Eierstockkrebs.

Frische klinischen Proben von Eierstockkrebs werden nach der Operation gesammelt (Abbildung 1) und in kleine Stücke (2A und 2B), die Zellaufschlämmung bilden geschnitten. Dies erlaubt eine optimale Belichtung der Proben in der enzymatischen Behandlung. Zelle Aufschlämmung einer enzymatischen Verdauung ausgesetzt und bei 37 ° C für 30 min inkubiert. Während der Inkubationszeit der Schlamm wird zunehmend bewölkt (vor allem nach jeder Bewegung), und dies ist ein Zei...

Watch this Scientific Journal Video about Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens at JoVE.com

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Diese Studie beschreibt eine detaillierte Methode zur Isolierung und Charakterisierung von primären Eierstockkrebszellen aus soliden klinischen Proben. Eierstockkrebs klinischen Proben werden enzymatische Verdauung unterzogen, um lebensfähig, Fibroblasten frei epithelialen Eierstockkrebs (EOC) Zellen sehr gut für Downstream-Anwendungen zu erhalten.

Verfahren zur Gewinnung von Primäreierstockkrebszellen von Solid Proben

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

method-for-obtaining-primary-ovarian-cancer-cells-from-solid-specimens

Research

JoVE Journal

Medicine

13.4K Aufrufe.  University of Minnesota.  Diese Studie beschreibt eine detaillierte Methode zur Isolierung und Charakterisierung von primären Eierstockkrebszellen aus soliden klinischen Proben. Eierstockkrebs klinischen Proben werden enzymatische Verdauung unterzogen, um lebensfähig, Fibroblasten frei epithelialen Eierstockkrebs (EOC) Zellen sehr gut für Downstream-Anwendungen zu erhalten. 

Serie: Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Verfahren zur Novel Anti-Cancer Drug Development mit Tumor Explantate von chirurgischen Proben


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Forschung

Medizin

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 ...

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Eine empfindliche Methode zur Quantifizierung Seneszente Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

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 ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Zwei Methoden zur Festlegung von primären humanen Endometrium Stromazellen aus Hysterektomie Proben

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Beurteilung von Eierstockkrebs Sphäroid Bindung und Invasion von Mesothelzellen in Echtzeit

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Mausmodell für nicht-invasive Bildgebungs zu erfassen und zu überwachen Eierstockkrebs Rezidiv

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Bereicherung für chemoresistenten Eierstockkrebs Stammzellen aus menschlichen Zelllinien

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

Quantitative massenspektrometrische Analyse von Krebs-Zellen-Proteome Abgeleitet von Liquid und soliden Tumoren

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

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 ...