Ringraziamo il SF 4206 ICORE (Université de Caen) per l&#39;utilizzo della struttura di citometria. JC ha ricevuto borse di studio post-dottorato dal Conseil de Radioprotezione d&#39;EDF e Conseil Régional della Bassa Normandia. Tali dati erano parte di progetti finanziati dalla Ligue Nationale contre le Cancer - Comités de l&#39;Orne et du Calvados.

1. Preparazione di C12FDG, bafilomicina A1, e Cell Culture <ol><li> Sciogliere la polvere C 12 FDG in dimetilsolfossido (DMSO) ad una concentrazione finale di 20 mM. Aliquota e conservare a -20 ° C. DMSO deve essere usato con adeguate condizioni di sicurezza. </li><li> Sciogliere la bafilomicina A1 polvere in DMSO ad una concentrazione finale di 0,1 mM. Aliquota e conservare a - 20 ° C. Bafilomicina deve essere usato con adeguate condizioni di sicurezza. </li><li> Coltivare RPMI 8226 linea cellulare, o altre linee cellulari aderenti o meno, in terreni contenenti 10% di siero fetale bovino e antibiotici 1%, a 37 ° C, 5% di CO 2. Stimare il numero di cellule necessarie per l&#39;esperimento. Per registrare un numero sufficiente di eventi durante l&#39;analisi di citometria di flusso, sono richiesti 5 x 10 5 cellule. Per determinare la percentuale di cellule senescenti, sono necessari tre campioni di cellule: campione senza etichetta, cellule non trattate colorate con C 12 FDG, cellule trattate colorate conC 12 FDG. </li><li> Il seme cellule 24 ore prima eseguendo l&#39;esperimento in modo che le cellule sono nel log-fase della curva di proliferazione cellulare. </li></ol> 2. L&#39;induzione di senescenza <ol><li> Indurre la senescenza delle cellule tumorali con l&#39;aggiunta di un farmaco chemioterapico. Concentrazione del farmaco e la durata del trattamento devono essere adattate al tipo di cellula testato. Iniziare con un trattamento di 48 ore alla concentrazione finale di 50 nM doxorubicina per una concentrazione di cellule a circa 10 6 cellule / ml. Non dimenticare di includere campione non trattato (controllo negativo). </li></ol> 3. Bafilomicina A1 Trattamento <ol><li> Controllare vitalità cellulare dalle cellule mescolando con trypan blue (v / v), come il farmaco chemioterapico utilizzato nel passaggio precedente potrebbe portare ad apoptosi. Riempire in una camera di Malassez e contare il numero di cellule blu (cellule morte) e di cellule bianche (cellule viventi). Se la percentuale di vitalità è inferiore al 70%, le cellule morte potrebbero essere removed utilizzando un kit appropriato per garantire che le cellule morte non sarà di preferenza la successiva analisi. </li><li> Rimuovere i terreni di coltura e di sostituire dai terreni di coltura fresca preriscaldata. Trattare le cellule con una concentrazione finale di 100 nM di bafilomicina A1. Bafilomicina A1 è usata per neutralizzare il pH acido dei lisosomi. Incubare per 1 ora a 37 ° C, 5% di CO 2. </li></ol> 4. C 12 FDG colorazione <ol><li> Sciogliere C 12 FDG preriscaldata in terreni di coltura fresco ad una concentrazione finale di 2 mm. </li><li> Aggiungere il composto di cellule ad una concentrazione finale di 33 mM e incubare 2 ore a 37 ° C, 5% di CO 2. Se utilizza cellule non aderenti, centrifugare le cellule a 300 xg per 5 minuti a 4 ° C (la velocità potrebbe dover essere adattato al tipo cellulare utilizzato), lavare 2x con PBS. Risospendere il pellet di cellule in 500 microlitri di PBS freddo. Se si utilizza cellule aderenti, rimuovere il supporto e lavare 2x con PBS. Raccogliere le cellule aderenti utilizzando una soluzione di tripsina e centrifugarecellule a 300 xg per 5 minuti a 4 ° C (la velocità potrebbe dover essere adattato al tipo cellulare utilizzato). Risospendere il pellet di cellule in 500 microlitri di PBS freddo. In entrambi i casi, la concentrazione cellulare dovrebbe essere di circa 10 6 cellule / ml. </li><li> Eseguire un co-immunomarcatura per caratterizzare ulteriormente la popolazione di cellule senescenti. </li></ol> 5. Citometria a Flusso Analisi <ol><li> Calibrare il citofluorimetro utilizzando le sfere di controllo raccomandate dal produttore del citometro a flusso. Per tutti i passi, almeno 10 4 eventi devono essere registrati. </li><li> Eseguire il primo campione contenente cellule senza etichetta. Preparare una a due parametri grafico visualizzazione Canale Forward Scatter (FSC) rispetto a canale laterale Scatter (SSC). Impostare i tubi fotomoltiplicatori (PMT) e definire una regione di interesse escludendo le cellule morte e detriti cellulari che potrebbero apparsi durante la preparazione del campione. Cancello questa regione di interesse in un istogramma a un parametro visualizza FL1 sulla scala logaritmicadella asse x ed il numero di eventi nel y. Il numero totale di eventi rappresenta il numero di cellule analizzate. Impostare il PMT in modo che le cellule senza etichetta compaiono nella prima decade su scala log del l&#39;asse x. Determinare l&#39;autofluorescenza delle celle, se necessario. </li><li> Eseguire il secondo campione contenente cellule non trattate. Sul istogramma a un parametro visualizzazione FL1 (fluorescenza di C 12 FDG), posizionare il cursore dopo la popolazione scarsamente etichettati. Questa soglia consente la discriminazione tra cellule non senescenti (cellule scarsamente etichettati) e le cellule senescenti (cellule brillantemente etichettati). </li><li> Esegui il terzo campione contenente cellule trattate. Brillantemente etichettato cellule, cioè le cellule senescenti appaiono sopra la soglia determinata nel passaggio precedente. Valutare la percentuale di cellule senescenti dividendo il numero di eventi luminosi per il numero totale di eventi. Se necessario, l&#39;attività della β-galattosidasi può anche essere stimata utilizzando la fluorescenza mediaintensità. </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>

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Abbiamo presentato qui un metodo di citometria a base di flusso per quantificare la senescenza cellulare in cellule tumorali dal vivo. Questo metodo si basa sull&#39;uso del composto membrana permeabile, il C 12 FDG, e può quindi essere utilizzato in qualsiasi tipi cellulari e dopo vari trattamenti finché il composto viene assorbito dalle cellule. Dopo l&#39;assorbimento cellulare, il C 12 FDG viene idrolizzato dalla β-galattosidasi, arricchito nei lisosomi delle cellule senescenti. Dopo eccitaz...

Poiché primi anni Sessanta e la sua scoperta da Hayflick e Moorhead, senescenza rimane una curiosità cellulare 1. Le cellule umane non proliferano indefinitamente e, da senescenza, Hayflick e Moorhead coniato il processo di invecchiamento cellulare. Senescenza è un arresto del ciclo cellulare stabile in cui le cellule rimangono metabolicamente attive in contrapposizione alla morte cellulare. Ad oggi, quattro meccanismi cellulari hanno mostrato di indurre senescenza: telomeri, danno al DNA, cromatina perturbazione, e anormale espressione di oncogeni mitogenico 2. Ciò indica che non solo le cellule normali, ma anche le cellule tumorali sono in grado di sottoporsi a senescenza 3. È un dato di fatto, le cellule di cancro sottoposti senescenza hanno mostrato di costituire una barriera ulteriore progressione tumorale 4-5. Tuttavia, diversi studi hanno ormai evidenziato che, in determinate circostanze, la senescenza cellulare può anche promuovere malignità 6-7. Studiare la senescenza di cancro celLS sta per diventare uno stimolo nella ricerca sul cancro come attualmente utilizzati farmaci chemioterapici possono portare alla senescenza delle cellule tumorali non solo in vitro ma anche in vivo 8. Il saggio maestro di indagare per la presenza di cellule senescenti è il saggio β-galattosidasi a pH acido. Questo saggio istochimico riflette l&#39;aumento nella biogenesi lisosomiale che si verificano nella maggior parte delle cellule senescenti. Brevemente, esogeno X-gal, applicato alle celle, è tagliata da β-galattosidasi arricchito in lisosomi delle cellule senescenti, dando origine ad un colore blu 9. Le cellule senescenti blu possono quindi essere quantificati al microscopio in campo chiaro. Anche se molto popolare, il saggio di β-galattosidasi presenta grossi inconvenienti. Prima, questa prova viene eseguita su cellule fissate. Secondo, è molto tempo perché implica per quantificare il grado di senescenza contando un significativamente elevato numero di cellule senescenti al microscopio. Terzo, questo dosaggionon è sensibile come la discriminazione tra cellule senescenti e non senescenti si basa interamente sul osservatore. Discutiamo qui un, analisi di citometria a base di veloce, sensibile e non sfruttato per valutare la presenza di cellule senescenti dal vivo. Questo saggio è basato sulla idrolisi di una membrana permeabile molecola, il 5-dodecanoylaminofluorescein di-β-D-galattopiranoside (C 12 FDG), da β-galattosidasi arricchita in cellule senescenti. Dopo idrolisi e eccitazione laser, il C 12 FDG emette fluorescenza verde e può quindi essere rilevata mediante citometria di flusso 10, 11. Il rilevamento di cellule senescenti tramite citofluorimetria offre il grande vantaggio di essere rapido e sensibile. Inoltre, la rilevazione di cellule senescenti mediante citometria di flusso può essere accoppiato con il rilevamento di altri marcatori cellulari. Questo test può essere utilizzato per rilevare le cellule senescenti all&#39;interno di una popolazione di cellule cancerose trattati con chemioterapia e può essere impostato routinariamentesu su sezioni di tessuto, dopo la dissociazione cellulare, in clinica.

<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

Le cellule umane non proliferano indefinitamente. Su indicazioni esterne e / o intrinseca, le cellule potrebbero morire o entrare in un arresto del ciclo cellulare stabile chiamato senescenza. Diversi meccanismi cellulari, come accorciamento dei telomeri e anormale espressione di oncogeni mitogeni, hanno dimostrato di causare senescenza. La senescenza non è limitato alle cellule normali, sono state riportate anche le cellule tumorali a senesce. Farmaci chemioterapici hanno dimostrato di indurre la senescenza nelle cellule tumorali. Tuttavia, resta controverso se la senescenza impedisce o promuove la tumorigenesi. Come potrebbe eventualmente essere paziente-specifico, sarà presto necessario un metodo rapido e sensibile per valutare senescenza in cellule di cancro. A tal fine, il saggio β-galattosidasi standard, il metodo attualmente utilizzato, presenta notevoli svantaggi: è tempo e non è sensibile. Proponiamo qui un saggio di citometria a base di flusso per studiare senescenza su Live cellule. Questo saggio offre il vantaggio di essere rapida, sensibile, e può essere accoppiato al immunomarcatura dei diversi marcatori cellulari.

Mieloma multiplo, un tumore ematologico, è stato utilizzato per valutare la senescenza indotta da una procedura di chemioterapici. Per fare così un linea cellulare MM commercialmente disponibile (RPMI 8226) è stato trattato per 2 giorni con doxorubicina, un farmaco chemioterapico. Le cellule sono state poi sottoposte a trattamento bafilomicina A1 e ulteriormente incubati con C 12 FDG. Le cellule sono state analizzate mediante citometria di flusso. Figura 1 illustra le diverse fasi e mostra...

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

Sia senescenza impedisce o promuove la tumorigenesi rimane controverso. Poiché i farmaci chemioterapici possono indurre le cellule tumorali a senesce, studiando la senescenza è essenziale per proporre nuove terapie. Tuttavia, il dosaggio standard di β-galattosidasi e ampiamente utilizzato presenta gravi inconvenienti. Proponiamo qui un saggio di citometria di flusso basato rapido e sensibile per quantificare senescenza.

Un metodo sensibile per quantificare le cellule tumorali senescenti

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

20.6K Views.  Université de Caen Basse-Normandie. Sia senescenza impedisce o promuove la tumorigenesi rimane controverso. Poiché i farmaci chemioterapici possono indurre le cellule tumorali a senesce, studiando la senescenza è essenziale per proporre nuove terapie. Tuttavia, il dosaggio standard di β-galattosidasi e ampiamente utilizzato presenta gravi inconvenienti. Proponiamo qui un saggio di citometria di flusso basato rapido e sensibile per quantificare senescenza.

Video: 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.

Un metodo per isolamento Islet murini e trapianto renale subcapsulare

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

Ricerca

Medicina

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.

Microscopia corneale confocale: A Novel tecnica non invasiva per quantificare Piccole Fibre Patologia neuropatie periferiche

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.

Un tempo reale impedenza elettrica tecnica basata su Misura Invasione di cellule endoteliali monostrato dalle cellule tumorali

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.

Un semplice metodo vite Guida per intracranica Studi dello xenotrapianto in topi

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.

Tre Culture dimensionali: strumento per studiare normale acinose Architettura vs trasformazione maligna delle cellule mammarie

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.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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.

Metodo per ottenere primarie cellule tumorali ovariche da campioni solidi

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.

Un<em&gt; In Vitro</em&gt; Dormienza Modello di estrogeno-sensibili cancro al seno nel midollo osseo: Uno strumento per gli studi molecolari del meccanismo e ipotesi 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 ...

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.

Utilizzo del saggio soft Agar Colony Formazione per identificare inibitori di tumorigenicità in cellule di carcinoma mammario

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.

Un metodo sensibile per quantificare le cellule tumorali senescenti

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