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1. Preparazione di vetrini di vetro per PAA <ol><li> Pulire 12 millimetri coprioggetto con panni bassi lint. </li><li> Flame i coprioggetti 12 mm e il coprioggetto 14 millimetri nel micropozzetto di ogni piatto di vetro fondo 35 millimetri facendoli passare attraverso una fiamma becco Bunsen con una pinzetta. </li><li> Trattare i pozzetti con 200 ml di 0,1 N NaOH per 5 min a temperatura ambiente. </li><li> Aspirare e asciugare i pozzetti per 30 min. </li><li> Trattare i pozzetti con 50-100 ml di 3-amminopropiltrimetossisilano per 10 minuti a temperatura ambiente nella cappa. Questo prodotto chimico reagisce con plastica; quindi, utilizzare pipette di vetro e non riempire completamente i pozzetti per evitare il contatto con la plastica piatto. </li><li> Lavare i pozzetti con acqua ultrapura per circa 10 minuti fino a quando il 3 amminopropiltrimetossisilano diventa chiaro. </li><li> Sciacquare i pozzetti con acqua ultrapura due volte con una bottiglia a spruzzo. </li><li> Lavare i pozzetti con 2 ml di acqua ultrapura unt temperatura ambiente su un bilanciere fissato a una velocità media (~ 1 Hz) per 10 min. </li><li> Aspirare e asciugare i pozzetti per 30 min. </li><li> Trattare i pozzetti con 2 ml di soluzione di glutaraldeide allo 0,5% a temperatura ambiente su un bilanciere fissato a una velocità media (~ 1 Hz) per 30 min. </li><li> Lavare i micropozzetti con 2 ml di acqua ultrapura a temperatura ambiente su un bilanciere funzionante a velocità media (~ 1 Hz) per 10 min. Ripetere altre due volte per un totale di 30 min. </li><li> Pozzetti a secco con un angolo ripido (60 ° o superiore) per 30 minuti. Nota: I piatti possono essere conservati per 2 mesi in un essiccatore. </li></ol> 2. Preparazione del PAA per Invadopodi saggi <ol><li> Per una soluzione del PAA molle (8% acrilammide e 0,05% BRI) 1 ml, 200 ml di combinare 40% acrilammide, 25 ml di 2% BIS, e 574 ml di acqua ultrapura (Tabella 1). </li><li> Per una soluzione del PAA disco (8% acrilammide e 0,35% BRI) 1 ml, 200 ml di combinare 40% acrilammide, 175 e #181; l del 2% BIS, e 409 ml di acqua ultrapura (Tabella 1). </li><li> Per una soluzione del PAA rigida (12% acrilammide e 0,60% BRI) 1 ml, 300 ml di combinare 40% acrilammide, 300 ml di 2% BIS, e 169 ml di acqua ultrapura (Tabella 1). </li><li> Degassare le soluzioni per 15 min. Aggiungere 200, 215, e 230 ml di 1 mg / ml di fibronectina plasma umano in acqua ultrapura alle molli, duri, rigidi e soluzioni di PAA, rispettivamente. Per tutte le soluzioni, aggiungere 1 ml di 10 mg / ml di acido acrilico N-idrossisuccinimmide (NHS) estere, 5 microlitri di 100 mg / ml, persolfato di ammonio e 2 ml di N, N, N &#39;, N&#39;-tetrametiletilendiammina (TEMED Tabella 1). Mescolare con dolce pipettaggio e evitare di creare bolle in soluzioni. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> PAA (1 ml) </td><td> 40% AA (Ml) </td><td> 2% (Ml) </td><td> Ultrapure Acqua (Ml) </td><td> 1 mg / ml FN (Ml) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 mg / ml NHS Ester (Ml) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 mg / ml APS (Ml) </td><td> TEMED (Ml) </td><td> Elastico Modulo (Pa) </td></tr><tr><td> Morbido </td><td> 200 </td><td> 25 </td><td> 574 </td><td> 200 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 1023 </td></tr><tr><td> Duro </td><td> 200 </td><td> 175 </td><td> 409 </td><td> 215 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 7307 </td></tr><tr><td> Rigido </td><td> 300 </td><td> 300 </td><td> 169 </td><td> 230 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 22692 </td></tr></tbody></table> Tabella 1: Ingrediente voluMES e conseguente moduli elastici per ZAP morbide, dure e rigide utilizzati nei test Invadopodi. AA = acrilamide, FN = fibronectina, APS = persolfato di ammonio. <ol start="5"><li> Pipettare 8,48 ml di soluzione di PAA al centro di ciascun pozzetto. Nota: Questo volume produce teoricamente PAA di 75 micron di spessore. </li><li> Abbassare delicatamente il lato fiammato del vetrino 12 mm appoggiata al droplet del micropozzetto con una pinzetta. </li><li> Lasciare che la soluzione PAA sandwich per polimerizzare per 15-30 min. Verificare polimerizzazione controllando la soluzione rimanente. </li><li> Aggiungere 2 ml di PBS 1x per ogni piatto e rimuovere i 12 millimetri coprioggetto con una pinzetta. Nota: 10x PBS viene effettuata in laboratorio e composto da 0,011 M KH 2 PO 4, 1,54 M di NaCl, e 0,056 M Na 2 HPO 4. </li><li> Lavare i pozzetti con 2 ml di 1x PBS a temperatura ambiente per 5 min. Ripetere altre due volte per un totale di 15 min. </li></ol> 3. Priparazione del PAA per forza trazione saggi <ol><li> Per una soluzione del PAA molle (8% acrilammide e 0,05% BRI) 1 ml, 200 ml di combinare 40% acrilammide, 25 ml di 2% BIS, e 566 ml di acqua ultrapura (Tabella 2). </li><li> Per una soluzione del PAA disco (8% acrilammide e 0,35% BRI) 1 ml, 200 ml di combinare 40% acrilammide, 175 ml di 2% BIS, e 401 ml di acqua ultrapura (Tabella 2). </li><li> Per una soluzione del PAA rigida (12% acrilammide e 0,60% BRI) 1 ml, 300 ml di combinare 40% acrilammide, 300 ml di 2% BIS, e 161 ml di acqua ultrapura (Tabella 2). </li><li> Degassare le soluzioni per 15 min. Aggiungere 200, 215, e 230 ml di 1 mg / ml di fibronectina plasma umano ai morbidi, duri, rigidi e soluzioni di PAA, rispettivamente. Sonicare 8 ml di 200 nm microsfere rosse fluorescenti (eccitazione / emissione di 580/605 nm) per 30 sec. Per ogni soluzione, aggiungere 8 ml di 200 microsfere fluorescenti rossi nm,1 ml di 10 mg / ml di acido acrilico estere NHS, 5 microlitri di 100 mg / persolfato di ammonio ml e 2 microlitri di TEMED (Tabella 2). Mescolare con dolce pipettaggio e evitare di creare bolle in soluzioni. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td style="width:40px"> PAA (1 ml) </td><td> 40% AA (Ml) </td><td> 2% BIS (Ml) </td><td style="padding-left: 4px; padding-right: 4px;"> Ultrapure Acqua (Ml) </td><td> 1 mg / ml FN (Ml) </td><td> Microsfere (Ml) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 mg / ml NHS Ester (Ml) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 mg / ml APS (Ml) </td><td> TEMED (Ml) </td><td> Elastico Modulo (Pa) </td></tr><tr><td> Morbido </td><td> 200 </td><td> 25 </td><td> 566 </td><td> 200 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 1023 </td></tr><tr><td> Duro </td><td> 200 </td><td> 175 </td><td> 401 </td><td> 215 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 7307 </td></tr><tr><td> Rigido </td><td> 300 </td><td> 300 </td><td> 161 </td><td> 230 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 22692 </td></tr></tbody></table> Tabella 2:. Volumi ingrediente e conseguente moduli elastici per ZAP morbide, dure e rigide utilizzati nei test di forza di trazione AA = acrilamide, FN = fibronectina, APS = persolfato di ammonio. <ol start="5"><li> Pipettare 8,48 ml di soluzione di PAA al centro di ciascun pozzetto. Nota: Questo volume produce teoricamente PAA di 75 micron di spessore. </li><li> Abbassare delicatamente il lato fiammata di the 12 millimetri coprioggetto al droplet del micropozzetto con una pinzetta. </li><li> Lasciare che la soluzione PAA sandwich per polimerizzare per 15-30 min. Verificare polimerizzazione controllando soluzione rimanente. </li><li> Aggiungere 2 ml di PBS 1x per ogni piatto e rimuovere i 12 millimetri coprioggetto con una pinzetta. </li><li> Lavare i pozzetti con 2 ml di 1x PBS a temperatura ambiente per 5 min. Ripetere altre due volte per un totale di 15 min. </li></ol> 4. Preparazione di ECM per Invadopodi saggi <ol><li> Riscaldare la soluzione di gelatina (1% gelatina e 1% di saccarosio) a 37 ° C. </li><li> Trattare le PAA con 150 microlitri della soluzione di gelatina per 1 min poi accuratamente aspirato dal fondo dei pozzetti quando si tiene i piatti fondo di vetro con un angolo di 45 °. </li><li> Essiccare il sottile strato residuo di gelatina sulle PAA ai pozzetti con un angolo ripido (60 ° o maggiore) per ottimizzare l&#39;essiccazione per 60 min. </li><li> Trattare i pozzetti con 2 ml di una soluzione di glutaraldeide allo 0,5% in refrigerate iCe per 15 minuti seguita da temperatura ambiente per 30 min. </li><li> Lavare i pozzetti con 2 ml di 1x PBS per 5 min. Ripetere altre due volte per un totale di 15 min. </li><li> Trattare i pozzetti con 2 ml di una soluzione di boroidruro di sodio per 1 min. Battere delicatamente piatti sul banco per rimuovere le bolle che si formano sulla superficie della gelatina. </li><li> Aspirare e lavare i pozzetti con 2 ml di 1x PBS per 5 min. Ripetere altre due volte per un totale di 15 min. </li><li> Diluire una soluzione fibronectina plasma umano marcato con FITC a 50 pg / ml con PBS 1x e centrifugare a 175.000 xg a 4 ° C per 15 min. O acquistare in commercio fibronectina etichettati o etichettarlo come segue: <ol><li> Disciogliere 5 mg di fibronectina in 10 ml di tampone borato (170 mM di sodio tetraidrato metaborato e 40 mM NaCl) e posto in tubi di dialisi. </li><li> Etichetta la fibronectina posizionando il tubo in 200 ml di tampone borato contenente 6 mg di FITC disciolto a temperatura ambiente su un agitatore magneticoimpostare al minimo per 1,5 ore al buio. </li><li> Dializzare con 500 ml di PBS 1x su un agitatore magnetico impostato al minimo per 3 giorni in una stanza buia fredda (4 ° C) con due volumi cambia un giorno. </li><li> Concentrazione fibronectina Calcolare FITC-marcato sulla base della densità ottica di aliquote diluito (1: 200) a 280 nm e 493 nm come [OD 280 - (0,36 x OD 493)] / 1,4 volte il fattore di diluizione di 200 volte 2 (che rappresenta per il glicerolo concentrare la fibronectina nel passaggio successivo) conseguente unità mg / ml. </li><li> Dializzare notte in una soluzione di glicerolo al 50% su un agitatore magnetico impostato al minimo durante la notte in una camera fredda buio a 4 ° C.) </li></ol></li><li> Trattare i pozzetti con 150 microlitri della soluzione di fibronectina FITC-marcato a temperatura ambiente per 60 minuti al buio. </li><li> Aspirare accuratamente la soluzione fibronectina coniugati con fluoresceina dal fondo dei pozzetti quando si tiene i piatti fondo di vetro aun angolo di 45 ° e quindi riempire il vetro piatti inferiori con soluzione di etanolo al 70% a temperatura ambiente per 10 min in una cappa di coltura cellulare scuro per la sterilizzazione. Anche riempire vetro fondo coperchi piatto o pulire con panni imbevuti lint bassi nel 70% di etanolo. </li><li> Lavare i piatti fondo di vetro da riempire con 1x PBS per 5 min. Ripetere altre due volte con 2 ml di PBS 1x per un totale di 15 min. </li></ol> 5. Preparazione e Imaging di Invadopodi Assays <ol><li> Aggiungere 25.000 cellule tumorali in 2 ml di terreno Invadopodi (1: 1 DMEM e RPMI 1640 con 5% di siero a basso contenuto proteico, siero bovino fetale al 10%, e 20 ng / ml di fresco aggiunto del fattore di crescita epidermico) ai piatti fondo di vetro e incubare una notte. </li><li> Aspirare e trattare i pozzetti con 2 ml di una soluzione di paraformaldeide 3,7% a temperatura ambiente al buio per 20 minuti per fissare le cellule. </li><li> Dopo due lavaggi rapidi con 2 ml di 1x PBS, trattare i pozzetti con 2 ml di Triton X-100 soluzione 0,1% a camera temperatura al buio per 5 min a permeabilize cellule. </li><li> Dopo un lavaggio rapido con 2 ml di 1x PBS, trattare i pozzetti con 3% di albumina sierica bovina soluzione bloccante a temperatura ambiente al buio per 60 minuti per bloccare le cellule. </li><li> Incubare le micropozzetti con 150 ml di anticorpo anti-topo cortactin (1: 750) in soluzione a temperatura ambiente al buio blocco per 60 min. </li><li> Lavare pozzetti con 2 ml di 1x PBS per 5 min. Ripetere altre due volte per un totale di 15 min. </li><li> Incubare i pozzetti con 150 ml di capra anti-topo 633 anticorpi (1: 500) e 546 falloidina (1: 750) in soluzione a temperatura ambiente al buio blocco per 60 min. </li><li> Lavare pozzetti con 2 ml di 1x PBS per 5 min. Ripetere altre due volte per un totale di 15 min. </li><li> Aggiungere sei gocce di mezzo di montaggio per riempire ogni micropozzetto e coprire con 22 x 22 lamelle. </li><li> Immagine cortactin e actina per identificare Invadopodi con eccitazione e di emissione filtri di 632/647 nm e556/570 nm, rispettivamente, con un obiettivo ad immersione in olio ad alta NA, 40X su un ampio campo di microscopio invertito. Analogamente, immagine fibronectina identificare degrado ECM utilizzando un filtro di eccitazione ed emissione di 492/518 nm. </li></ol> 6. Preparazione e Imaging di forza trazione Assays <ol><li> Aggiungere 15.000 cellule tumorali in 2 ml di terreno Invadopodi ai piatti fondo di vetro e incubare durante la notte. </li><li> Aspirare media nei piatti fondo di vetro e sostituire con 2 ml di mezzo L-15, con le stesse integratori (siero ipoproteica 5%, siero bovino fetale al 10%, e 20 ng / ml di fresco aggiunto del fattore di crescita epidermico), pre -warmed a 37 ° C. </li><li> Incubare i piatti inferiori vetro in una camera ambientale su un microscopio invertito pre-equilibrati a 37 ° C con umidità elevata per 1 ora. </li><li> Prendere quattro serie di immagini con un elevato NA, 40X obiettivo su un microscopio a grande campo invertito, con una fase automatizzata per marcare posizioni cellulari: <ol><li> Celle di immagine su dip dei PAA con contrasto di fase (immagine &quot;fase&quot;). </li><li> Microsfere fluorescenti immagine utilizzando filtri di eccitazione ed emissione di 560/645 nm concentrandosi leggermente sotto le celle fino al primo strato di microsfere nella parte superiore del PAA è a fuoco (immagine &quot;sottolineato&quot;). </li><li> In aggiunta, si concentrano al fondo del PAA (utilizzando le microsfere come marcatori) e prendere una immagine (un&#39;immagine &quot;bottom&quot;). Nota: Lo scopo di questa immagine è di registrare la z-posizione per ottenere lo spessore effettivo PAA in ciascuna posizione per i calcoli forza di trazione come descritto in Risultati rappresentativi. </li><li> Dopo queste immagini sono state acquisite a più posizioni, mescolare delicatamente in 220 ml di soluzione al 10% Triton-X per rimuovere le cellule. Microsfere fotografia in cima ZAP in ciascuna posizione come precedentemente descritto (immagine &quot;nullo&quot;). </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

Questo lavoro è stato sostenuto dal National Cancer Institute dei National Institutes of Health in premio Numero K25CA143412 (Parekh). Vorremmo riconoscere che un ulteriore sostegno è stato fornito dal Dipartimento di Otorinolaringoiatria. Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Institutes of Health.

Vi presentiamo un metodo per la fabbricazione di PAA che possono essere utilizzati come base per Invadopodi e forza di trazione saggi di correlare i comportamenti cellulari invasive e contrattili. Mentre CPA sono stati a lungo utilizzati per osservare gli effetti sulle cellule rigidità e calcolare le forze di trazione 18,24,27, questo protocollo è il primo a sviluppare test paralleli basati su CPA con le stesse rigidità di correlare i comportamenti cellulari invasive e contrattili in risposta alla matrice ...

La rigidità del ECM associati al tumore è stato identificato come un fattore significativo nel determinare comportamenti maligni aumentando actomiosina contrattilità 1-3. Anche se questo effetto è stato dimostrato soprattutto con le cellule del cancro al seno, la rigidità matrice è stato trovato per modificare le proprietà invasive di cellule derivate da una varietà di tumori 4-8 che suggeriscono che la rigidità del tumore può svolgere un ruolo in altri tipi di tumori. Per penetrare i tessuti reticolati durante la migrazione invasivo, le cellule tumorali utilizzano sporgenze adesive ricche di actina conosciuti come Invadopodi che localizzare proteasi a degradare focally ECM 9. Invadopodi sono considerati un segno distintivo di cellule invasive e sono stati implicati in invasione delle cellule tumorali e metastasi 10,11. Il lavoro precedente ha mostrato che la rigidità matrice può regolare numeri Invadopodi e associati ECM degrado 4,12 attraverso l&#39;attività miosina II e proteine ​​meccanosensibili 12. Dato checorrelazione tra la densità del tumore e cancro aggressività 13,14, questi risultati suggeriscono un meccanismo attraverso il quale le cellule tumorali possono rispondere ai tessuti tumorali rigidi guidare invasione e metastasi attraverso actomiosina contrattilità. In vitro rigidità ECM e nella densità del tessuto vivo hanno dimostrato di regolare il comportamento invasivo delle cellule tumorali 1,15-17. Mentre actomiosina contrattilità sembra essere importante in questo processo, gli studi in corso il conflitto come se la capacità metastatica è correlata ad un aumento o diminuzione forze contrattili 6,18-20. Inoltre, resta noto se queste forze mediare direttamente l&#39;attività Invadopodi 21. Abbiamo recentemente scoperto che le forze contrattili cellula tumorale dipendevano rigidità matrice ed erano predittivi di ECM degrado da Invadopodi 5. Questi risultati suggeriscono che le forze cellulari possono svolgere un ruolo importante nella progressione del cancro mediando Invadopodi actività in risposta alle proprietà meccaniche del microambiente tumorale. Al fine di correlare le proprietà invasive e contrattili delle cellule tumorali 5, abbiamo modificato un protocollo per la creazione di ZAP con differenti rigidità precedentemente utilizzati per studiare l&#39;attività Invadopodi rigidità-dipendente 4,12,22. Con reticolazione chimica fibronectina plasma umano durante ZAP, questi idrogel modificati possono essere utilizzate come base per entrambi Invadopodi e forza di trazione saggi per assicurare che le cellule sperimentato le stesse rigidità in entrambi gli esperimenti 5. Nei saggi Invadopodi, la fibronectina fornisce un dominio di legame naturale per la gelatina di collegare la ECM sovrapposto alla ZAP per rilevare degradazione della matrice. Nei saggi di forza di trazione, la fibronectina fornisce un ligando per adesione cellulare diretta per rilevare spostamenti microsfere utilizzate per calcolare le forze di trazione cellulari. Questo metodo comporta quello che abbiamo chiamato morbido, duro,e CPA rigidi che sono legati ai piatti fondo di vetro e hanno moduli elastici, E, di 1.023, 7.307, e 22.692 Pa 5 che abbracciano la gamma di proprietà meccaniche segnalate per i tessuti normali e tumorali 23.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-Aminopropyltrimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-Rad</td>
			<td>161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic acid NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>A8060</td>
			<td>prepare fresh in fume hood 10 mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Life Technologies</td>
			<td>A22283</td>
			<td>can also use rhodamine</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>prepare fresh 10% solution in 1X PBS</td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>18606</td>
			<td>use six drops to fill microwells</td>
		</tr>
		<tr>
			<td>BIS (2%)</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>RPI</td>
			<td>A30075</td>
			<td>make 3% for blocking solution in 1X PBS and store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips (12 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9777</td>
			<td>pre-equilibrate in borate buffer for 15-30 min</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>use to make acrylic acid NHS ester solution</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PHARMCO-AAPER</td>
			<td>E200</td>
			<td>dilute with ultrapure water to 70%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>SH30070.03</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Polysciences</td>
			<td>00639</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (35 mm coverslips)</td>
			<td>MatTek</td>
			<td>P35G-0-14-C</td>
			<td>coverslips are uncoated</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%)</td>
			<td>Polysciences</td>
			<td>01909</td>
			<td>dilute with 1X PBS to 0.5%</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa Fluor 633 antibody&#160;</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Life Technologies</td>
			<td>33016-015</td>
			<td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>PX-1565-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>mouse anti-cortactin 4F11 antibody&#160;</td>
			<td>EMD Millipore</td>
			<td>05-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>SX-0720-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23020</td>
			<td>use to make 10X PBS stock and borate buffer</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>dilute with ultrapure water to 0.1 N</td>
		</tr>
		<tr>
			<td>Nu-Serum (low-protein serum)</td>
			<td>BD Biosciences</td>
			<td>355500</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros</td>
			<td>416785000</td>
			<td>typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td>
		</tr>
		<tr>
			<td>PBS (sterile)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>use for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Cellgro</td>
			<td>10-040-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452882</td>
			<td>prepare fresh in fume hood 1 mg/ml in 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>sodium metaborate tetrahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0251</td>
			<td>use to make borate buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td>make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining</td>
		</tr>
	</tbody>
</table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

Tessuti tumorali rigidi sono stati fortemente implicati nella regolazione della migrazione delle cellule del cancro e l&#39;invasione. Migrazione invasiva attraverso i tessuti reticolati è facilitato da sporgenze ricchi di actina chiamati Invadopodi che proteoliticamente degradano la matrice extracellulare (ECM). Attività Invadopodi ha dimostrato di essere dipendente da rigidità ECM e il cancro delle cellule contrattili forze che suggeriscono che i segnali di rigidità possono regolare queste strutture subcellulari attraverso actomiosina contrattilità. Proprietà invasive e contrattili delle cellule tumorali possono essere correlati in vitro utilizzando Invadopodi e forza di trazione test basati su gel di poliacrilammide (PaaS) di diversa rigidità. Proprietà invasive e contrattili delle cellule tumorali possono essere correlati in vitro utilizzando Invadopodi e forza di trazione test basati su gel di poliacrilammide (PaaS) di diversa rigidità. Mentre esistono delle differenze tra i due test, il protocollo qui presentata fornisce un metodo per la creazione di PAA thin può essere utilizzato in entrambi i test e sono facilmente adattabili alle esigenze biologiche e tecniche specifiche dell&#39;utente.

Nel saggio Invadopodi, Invadopodi sono in genere identificato da colocalizzazione di marcatori come actina e cortactin a strutture puntiformi all&#39;interno del corpo cellulare (Figura 1). Sia attivamente degradante e non degradanti Invadopodi può essere contato e si differenziano per se queste strutture sono colocalized con aree nere prive segnale fluorescente nella fibronectina coniugati con fluoresceina (Figura 1). Invadopodi vengono contati manualmente, e la degradazione ECM per c...

Watch this Scientific Journal Video about Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells at JoVE.com

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigidità meccanica nel microambiente tumorale gioca un ruolo cruciale nel guidare il comportamento maligno aumentando l&#39;attività Invadopodi e actomiosina contrattilità. Utilizzando gel di poliacrilammide (PaaS), Invadopodi e forza di trazione test possono essere utilizzati per studiare le proprietà invasive e contrattili delle cellule tumorali in risposta alla matrice rigidità.

Gel di poliacrilammide per Invadopodi e trazione Forza saggi sulle cellule tumorali

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

polyacrylamide-gels-for-invadopodia-traction-force-assays-on-cancer

Research

JoVE Journal

Medicine

12.0K Views.  Vanderbilt University Medical Center.  Rigidità meccanica nel microambiente tumorale gioca un ruolo cruciale nel guidare il comportamento maligno aumentando l'attività Invadopodi e actomiosina contrattilità. Utilizzando gel di poliacrilammide (PaaS), Invadopodi e forza di trazione test possono essere utilizzati per studiare le proprietà invasive e contrattili delle cellule tumorali in risposta alla matrice rigidità. 

Video: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Lavorazione dei tessuti primaria tumore al cervello per saggi su cellule staminali e flusso di Ordinamento

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Ricerca

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Formazione di epitelio prostatico umano utilizzando tessuti ricombinazione dei roditori urogenitale Mesenchima Sinus e cellule staminali umane

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adattamento del semiautomatico circolanti (CTC) Saggi Tumor Cell per applicazioni cliniche e di ricerca preclinica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling di microRNA regolati dagli estrogeni in cellule di carcinoma mammario

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

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 ...

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.

Arricchimento per chemioresistente Ovarian Cancer Stem Cells da linee cellulari umane

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

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.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Un sistema organotipica High Throughput per la caratterizzazione di droga sensibilità di primaria multicampo cellule del mieloma

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...