Wir danken dem SF 4206 ICORE (Université de Caen) für die Nutzung der Durchflusszytometrie Anlage. JC erhielt Post-Doc-Stipendien aus Conseil de Strahlenschutz d&#39;EDF und Conseil Régional der Basse-Normandie. Diese Daten wurden im Rahmen von Projekten von der Ligue Nationale contre le Cancer finanziert - Comités de l&#39;Orne et du Calvados.

1. Herstellung von C12FDG, Bafilomycin A1 und Zellkultur <ol><li> Man löst den C 12 FDG Pulver in Dimethylsulfoxid (DMSO) zu einer Endkonzentration von 20 mM. Aliquot und bei -20 ° C DMSO sollte mit entsprechenden Sicherheits-Bedingungen verwendet werden. </li><li> Man löst den bafilomycin A1 Pulver in DMSO auf eine Endkonzentration von 0,1 mM. Aliquot und lagern bei - 20 ° C. Bafilomycin sollte mit entsprechenden Sicherheits-Bedingungen verwendet werden. </li><li> Kultivieren RPMI 8226-Zellinie, oder andere Zelllinien haftende oder nicht, in Medium, enthaltend 10% fötales Kälberserum und 1% Antibiotika, bei 37 ° C, 5% CO 2. Schätzen der Anzahl von Zellen für das Experiment benötigt. Um eine ausreichende Anzahl von Ereignissen in der Durchflusszytometrie aufgezeichnet werden 5 x 10 5 Zellen erforderlich. Um den Prozentsatz der alternden Zellen bestimmen, drei Zellproben erforderlich: für unmarkierte Proben, unbehandelte Zellen, die mit C 12 FDG gefärbt, behandelte Zellen angefärbt mitC 12 FDG. </li><li> Seed die Zellen 24 Stunden vor Durchführung des Experiments so dass die Zellen in der log-Phase der Zellproliferation Kurve sind. </li></ol> 2. Induktion von Seneszenz <ol><li> Induce Seneszenz von Krebszellen, indem Sie eine chemotherapeutische Medikament. Drug Konzentration und Dauer der Behandlung sollte der Zelltyp getestet angepasst werden. Beginnen mit einer 48-Stunden-Behandlung in einer Endkonzentration von 50 nM Doxorubicin für eine Konzentration der Zellen bei etwa 10 6 Zellen / ml. Vergessen Sie nicht, unbehandelten Probe sind (negative Kontrolle). </li></ol> 3. Bafilomycin A1 Behandlung <ol><li> Überprüfen Zelllebensfähigkeit durch Mischen von Zellen mit Trypanblau (v / v), die chemotherapeutische Medikament in dem vorhergehenden Schritt verwendet werden, um die Apoptose führen könnten. Füllen Sie in eine Kammer Malassez und zählen die Anzahl der blauen Zellen (tote Zellen) und der weißen Blutkörperchen (lebende Zellen). Wenn der Prozentsatz der Lebensfähigkeit ist weniger als 70%, vielleicht toten Zellen rem seinoved Verwendung eines geeigneten Kit, um sicherzustellen, dass tote Zellen nicht beeinflussen die nachfolgende Analyse. </li><li> Entfernen Sie die Kulturmedien und ersetzen durch vorgewärmte frische Kulturmedien. Behandeln der Zellen mit einer Endkonzentration von 100 nM bafilomycin A1. Bafilomycin A1 wird verwendet, um den sauren pH der Lysosomen zu neutralisieren. Inkubieren 1 h bei 37 ° C, 5% CO 2. </li></ol> 4. C 12 FDG Färbung <ol><li> Auflösen C 12 FDG in vorgewärmte frische Nährmedien in einer Endkonzentration von 2 mM. </li><li> In der Verbindung zu den Zellen mit einer Endkonzentration von 33 uM und inkubiert 2 h bei 37 ° C, 5% CO 2. Bei der Verwendung von nicht-haftenden Zellen zentrifugiert, die Zellen bei 300 xg für 5 min bei 4 ° C (die Geschwindigkeit haben könnte, um den verwendeten Zelltyp angepasst werden), 2x mit PBS waschen. Zellpellet in 500 ul kaltem PBS. Bei Verwendung von anhaftenden Zellen, entfernen Sie die Medien und waschen 2x mit PBS. Ernten Sie die anhaftenden Zellen unter Verwendung einer Trypsin-Lösung und ZentrifugeZellen bei 300 g für 5 min bei 4 ° C (die Geschwindigkeit möglicherweise zum verwendeten Zelltyp eingestellt werden). Zellpellet in 500 ul kaltem PBS. In beiden Fällen sollte die Zellkonzentration etwa 10 6 Zellen / ml. </li><li> Führen Sie eine Co-Immunmarkierung weiter zu charakterisieren, die Bevölkerung von alternden Zellen. </li></ol> 5. Durchflusszytometrie <ol><li> Kalibrieren des Durchflusszytometer unter Verwendung der Steuerung Perlen vom Hersteller des Durchflusszytometer empfohlen. Für alle Schritte müssen mindestens 10 4 Ereignisse aufgezeichnet werden sollen. </li><li> Führen Sie die erste Probe, die unmarkierten Zellen. Bereiten Sie eine Zwei-Parameter-Grafik Anzeige Forward Scatter Channel (FSC) gegen Side Scatter Channel (SSC). Stellen Sie die Photomultiplier Tubes (PMT) und definieren Sie einen Bereich von Interesse außer abgestorbenen Zellen und Zelltrümmer, die während der Probenvorbereitung erschien haben könnte. Tor diese Region von Interesse in einer Ein-Parameter-Histogramm, FL1 auf der logarithmischen Skalader x-Achse und die Anzahl von Ereignissen in der y-Achse. Die Gesamtzahl der Ereignisse die Anzahl der analysierten Zellen. Stellen Sie die PMT so dass unmarkierten Zellen in der ersten Dekade erscheinen auf der logarithmischen Skala der x-Achse. Bestimmen Sie die Autofluoreszenz der Zellen, wenn nötig. </li><li> Führen Sie die zweite Probe, die unbehandelten Zellen. Auf der Ein-Parameter-Histogramm, FL1 (Fluoreszenz von C 12 FDG), platzieren Sie den Cursor nach der schwach markierten Bevölkerung. Diese Schwelle wird es die Unterscheidung zwischen nicht-alternden Zellen (schwach markierten Zellen) und alternden Zellen (hell markierten Zellen). </li><li> Führen Sie die dritte Probe, die behandelten Zellen. Hell markierten Zellen scheinen also alternden Zellen über dem Schwellenwert in dem vorherigen Schritt bestimmt. Bewerten Sie den Prozentsatz der alternden Zellen, indem die Zahl der hellen Ereignisse pro Gesamtzahl der Veranstaltungen. Falls erforderlich, kann die Aktivität der β-Galactosidase auch unter Verwendung der mittleren Fluoreszenz werdenIntensität. </li></ol>

<ol>
	<li>Hayflick, L., Moorhead, P. 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+serial+cultivation+of+human+diploid+cell+strains.">The serial cultivation of human diploid cell strains.</a> Experimental Cell Research. 25, 585-621 (1961).</li><li>Campisi, J., d'Adda di Fagagna, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+when+bad+things+happen+to+good+cells.">Cellular senescence: when bad things happen to good cells.</a> Nature Review Molecular Cell Biology. 8 (9), 729-740 (2007).</li><li>Shay, J. W., Roninson, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+senescence+in+carcinogenesis+and+cancer+therapy.">Hallmarks of senescence in carcinogenesis and cancer therapy.</a> Oncogene. 23 (16), 2919-2933 (2004).</li><li>Bartkova, J., Rezaei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogene-induced+senescence+is+part+of+the+tumorigenesis+barrier+imposed+by+DNA+damage+checkpoints.">Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.</a> Nature. 444 (7119), 633-637 (2006).</li><li>Takacova, S., Slany, R., Bartkova, J., Stranecky, V., Dolezel, P., Luzna, P., 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=DNA+damage+response+and+inflammatory+signaling+limit+the+MLL-ENL-induced+leukemogenesis+in+vivo.">DNA damage response and inflammatory signaling limit the MLL-ENL-induced leukemogenesis in vivo.</a> Cancer Cell. 21, 517-531 (2012).</li><li>Krtolica, A., Parrinello, S., Lockett, S., Desprez, P. Y., Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescent+fibroblasts+promote+epithelial+cell+growth+and+tumorigenesis:+a+link+between+cancer+and+aging.">Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.</a> Proceedings of the National Academy of Sciences. 98, 12072-12077 (2001).</li><li>Coppe, J. P., Patil, C. K., Rodier, F., Sun, Y., Munoz, D. P., Goldstein, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS Biology. 6, 2853-2868 (2008).</li><li>Roninson, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+senescence+in+cancer+treatment.">Tumor cell senescence in cancer treatment.</a> Cancer Research. 63 (11), 2705-2715 (2003).</li><li>Dimri, G. P., Lee, X., 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=A+biomarker+that+identifies+senescent+human+cells+in+culture+and+in+aging+skin+in+vivo.">A biomarker that identifies senescent human cells in culture and in aging skin in vivo.</a> Proceedings of the National Academy of Sciences. 92 (20), 9363-937 (1995).</li><li>Kurz, D. J., Decary, S., Hong, Y., Erusalimsky, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+(beta)-galactosidase+reflects+an+increase+in+lysosomal+mass+during+replicative+ageing+of+human+endothelial+cells.">Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.</a> Journal of Cell Science. 113, 3613-3622 (2000).</li><li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+detect+senescence-associated+beta-galactosidase+(SA-betagal)+activity,+a+biomarker+of+senescent+cells+in+culture+and+in+vivo.">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nature Protocols. 4 (12), 1798-1806 (2009).</li><li>Cahu, J., Bustany, S., Sola, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+secretory+phenotype+favors+the+emergence+of+cancer+stem-like+cells.">Senescence-associated secretory phenotype favors the emergence of cancer stem-like cells.</a> Cell Death and Disease. Dec 20, 3-e446 (2012).</li></ol>

Keine Interessenskonflikte erklärt.

Wir präsentierten hier eine Durchflusszytometrie basierende Methode, um zelluläre Seneszenz in lebenden Krebszellen zu quantifizieren. Diese Methode beruht auf der Verwendung der Membran durchlässig Verbindung, die C 12-FDG Basis und kann daher in allen Zelltypen und nach verschiedenen Behandlungen verwendet werden, solange die Verbindung von den Zellen aufgenommen. Nach Aufnahme in die Zelle ist die C 12 FDG hydrolysiert durch die β-Galactosidase, in Lysosomen von alternden Zellen angereichert...

Seit Anfang der sechziger Jahre und seiner Entdeckung durch Hayflick und Moorhead, Seneszenz bleibt eine zelluläre Neugier 1. Menschliche Zellen nicht unbegrenzt vermehren und durch Seneszenz Hayflick und Moorhead den zellulären Alterungsprozess geprägt. Seneszenz ist eine stabile Arretierung des Zellzyklus in dem Zellen metabolisch aktiv bleiben, um Zelltod entgegen. Bisher wurden vier zellulären Mechanismen gezeigt, Seneszenz induzieren: Verkürzung der Telomere, DNA-Schäden, Chromatin Störung und abnormale Expression von Onkogenen mitogene 2. Dies deutet darauf hin, dass nicht nur normale Zellen, sondern auch Krebszellen sind in der Lage, Seneszenz 3 zu unterziehen. In der Tat wurden Krebszellen unterziehen Seneszenz gezeigt, um eine Barriere zur weiteren Tumorprogression 4-5 darstellen. Allerdings haben einige Berichte nun darauf hin, dass unter bestimmten Umständen, zelluläre Seneszenz kann auch die Förderung Bösartigkeit 6-7 hingewiesen. Studieren Seneszenz von Krebs cells ist im Begriff, ein Drang in der Krebsforschung als derzeit verwendeten Chemotherapeutika können Seneszenz von Krebszellen nicht nur in vitro, sondern auch in vivo 8 führen werden. Die Master-Assay, um das Vorhandensein von alternden Zellen zu untersuchen ist β-Galactosidase-Assay bei saurem pH-Wert. Diese histochemischen Test spiegelt die in lysosomalen Biogenese vorkommenden in den meisten alternden Zellen erhöht. Kurz gesagt, wird exogene X-gal, die auf Zellen von β-Galactosidase in Lysosomen von alternden Zellen angereichert gespalten, was zu einer blauen Farbe 9. Die blauen alternden Zellen können dann unter einem Hellfeld-Mikroskop quantifiziert werden. Obwohl sehr beliebt, stellt die β-Galactosidase Assay große Nachteile. Zuerst wird dieser Test auf fixierte Zellen durchgeführt. Zweitens ist es sehr zeitaufwendig, weil es um den Grad der Seneszenz durch Zählen eine signifikant hohe Anzahl von alternden Zellen unter einem Mikroskop zu quantifizieren impliziert. Drittens, dieser Testnicht empfindlich ist als die Unterscheidung zwischen alternden und nicht-alternden Zellen ganz auf die Betrachter. Wir diskutieren hier ein ungenutztes, schnell und empfindlich Durchflusszytometrie basierende Assay auf das Vorhandensein von lebenden alternden Zellen beurteilen. Dieser Assay auf der Hydrolyse von einer Membran durchlässig Molekül, das 5-dodecanoylaminofluorescein di-β-D-galactopyranosid (C 12 FDG) basiert, durch β-Galactosidase in alternden Zellen angereichert. Nach der Hydrolyse-und Laser-Anregung emittiert der C 12 FDG grüne Fluoreszenz und kann daher mittels Durchflusszytometrie 10, 11 detektiert werden. Der Nachweis von alternden Zellen via Durchflusszytometrie bietet den großen Vorteil, dass sie schneller und empfindlicher. Darüber hinaus kann die Erfassung von alternden Zellen mittels Durchflusszytometrie mit der Detektion von anderen zellulären Marker gekoppelt werden. Dieser Test kann verwendet werden, um alternde Zellen in einer Population von Krebszellen mit einer Chemotherapie behandelt werden, erkennen und kann routinemäßig eingestellt werdenauf Gewebeschnitten nach zellulären Dissoziation, in der Klinik.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>5-dodecanoylaminofluorescein di-&beta;-D-galactopyranoside </td>
			<td>Invitrogen</td>
			<td>D-2893</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dimethyl sulfoxide </td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bafilomycin A1</td>
			<td>Sigma</td>
			<td>B1793</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxorubicin</td>
			<td>Sandoz</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RPMI 1640 cell culture medium</td>
			<td>Lonza</td>
			<td>BE12-702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal calf serum</td>
			<td>PAA Laboratories GmBH</td>
			<td>A15 101</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotics</td>
			<td>Gibco</td>
			<td>5290-018</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gallios flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>A94291</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a sensitive method to quantify senescent cancer cells

Menschliche Zellen nicht unbegrenzt vermehren. Bei externen und / oder intrinsische Signale könnten Zellen sterben oder geben Sie einen stabilen Zellzyklus genannt Seneszenz. Mehrere zellulären Mechanismen, wie Telomerverkürzung und abnormale Expression mitogene Onkogene, haben gezeigt, dass Seneszenz bewirken. Seneszenz nicht auf normalen Zellen beschränkt; Krebszellen haben auch berichtet, altern. Chemotherapeutika wurde gezeigt, Seneszenz in Krebszellen zu induzieren. Es bleibt jedoch umstritten, ob Seneszenz verhindert oder fördert Tumorentstehung. Als es schließlich könnte Patienten-spezifischen, wird eine schnelle und empfindliche Methode zur Seneszenz in Krebszelle beurteilen bald erforderlich sein. Zu diesem Zweck stellt die Standard-β-Galactosidase-Assay wird die aktuell verwendete Methode, wesentliche Nachteile: es ist zeitaufwendig und nicht empfindlich. Wir schlagen hier eine Durchflusszytometrie-basierte Assays zur Seneszenz auf li studierenve Zellen. Dieser Test hat den Vorteil, dass eine schnelle, empfindliche und können zur Immunmarkierung von verschiedenen zellulären Marker gekoppelt werden.

Multiples Myelom, eine hämatologische Malignität, wurde verwendet, um das Altern von einem chemotherapeutischen Verfahren induziert bewerten. Dazu muss ein handelsübliches MM-Zelllinie (RPMI 8226) für 2 Tage mit Doxorubicin, einem chemotherapeutischen Medikament behandelt wurde. Die Zellen wurden dann bafilomycin A1 unterzogen und weiter mit C 12 FDG inkubiert. Die Zellen wurden mittels Durchflusszytometrie analysiert. Abbildung 1 zeigt die verschiedenen Schritte und zeigt, dass der Proze...

Watch this Scientific Journal Video about A Sensitive Method to Quantify Senescent Cancer Cells at JoVE.com

A Sensitive Method to Quantify Senescent Cancer Cells

Ob Seneszenz verhindert oder fördert Tumorentstehung bleibt umstritten. Da Chemotherapeutika Krebszellen veranlassen können, altern, Studium Seneszenz ist für Vorschläge für neue Therapien unerlässlich. Allerdings stellt die Standard-und im Großen und Ganzen verwendet β-Galactosidase Assay große Nachteile. Wir schlagen hier eine schnelle und empfindliche Durchflusszytometrie-basierte Assays zur Quantifizierung Seneszenz.

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. ...

a-sensitive-method-to-quantify-senescent-cancer-cells

Research

JoVE Journal

Medicine

20.5K Aufrufe.  Université de Caen Basse-Normandie. Ob Seneszenz verhindert oder fördert Tumorentstehung bleibt umstritten. Da Chemotherapeutika Krebszellen veranlassen können, altern, Studium Seneszenz ist für Vorschläge für neue Therapien unerlässlich. Allerdings stellt die Standard-und im Großen und Ganzen verwendet β-Galactosidase Assay große Nachteile. Wir schlagen hier eine schnelle und empfindliche Durchflusszytometrie-basierte Assays zur Quantifizierung Seneszenz.

Serie: A Sensitive Method to Quantify Senescent Cancer Cells

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Eine Methode zur Murine Islet Isolation und subkapsulären Nierentransplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Forschung

Medizin

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

Corneal konfokale Mikroskopie: A Novel Non-invasive Technik zu Small Fibre Pathologie in peripheren Neuropathien Quantify

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Eine Echtzeit-Elektrische Impedanz basierte Technik zur Invasion von Endothelzellen Monolayer von Krebs Zellen zu messen

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

Eine einfache Anleitung Screw Verfahren zur Intrakranielle Xenograft Studien an Mäusen

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Dreidimensional Kulturen: Ein Werkzeug, um Architektur zu studieren Normale acinar gegen maligne Transformation von Brustzellen

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. 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 ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

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 ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Ein<em&gt; In-vitro-</em&gt; Winterruhe Model of Östrogen-sensitiven Brustkrebs im Knochenmark: Ein Werkzeug für Molecular Mechanism Studien und Hypothesengenerierung

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Nutzung der Koloniebildung in Weichagar-Assay auf Inhibitoren der Tumorigenität in Brustkrebs-Zellen zu identifizieren

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

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.