This research is supported by funding from CBCF (to ZW).

1. Preparare le cellule per Immagini dal vivo <ol><li> Coltura cellulare e la semina <ol><li> Utilizzare le cellule MCF-7 di tumore mammario selezionate per la resistenza a docetaxel (MCF-7 TXT) e la loro linea cellulare dei genitori non resistente (MCF-7 CC). Il processo di selezione dettagliata e la caratterizzazione di queste linee cellulari selezionate sono state descritte in precedenza 24. </li><li> Grow tutte le cellule in coltura 10 cm a 37 ° C in un mezzo costituito 90% del mezzo di Dulbecco modificato Eagle (DMEM) e il 10% di siero fetale bovino (FBS) e integrati con amminoacidi non essenziali. Mantenere il mezzo in CO 2 atmosphere 5%. Mantenere le cellule MCF-7 TXT a 5 nM docetaxel. </li><li> cellule seme su vetrini per l&#39;imaging dal vivo. Sterilizzare 24 mm vetrini poli-L-lisina rivestite risciacquandole con il 70% di alcol e poi esporli ai raggi UV durante la notte. Mettere i coprioggetti in una piastra di coltura 6 pozzetti di 35 mm. Plasso un vetrino in ciascun pozzetto. </li><li> Rimuovere il mezzo dal piatto di coltura di 10 cm e lavare le cellule con PBS. Aggiungere 0,5 ml di tripsina-EDTA al piatto per staccare le cellule dal piatto. Aggiungere 5 ml di terreno di coltura al piatto di neutralizzare la tripsina dopo il distacco delle cellule. </li><li> Contare le cellule con un emocitometro per determinare il numero di cellule per unità di volume. </li><li> Aggiungere circa 10 5 cellule in ciascun pozzetto in base al numero di cellule per unità di volume. Dopo aver agitato delicatamente, mettere la piastra posteriore in incubatrice per permettere alle cellule di allegare alle lamelle. </li></ol></li><li> L&#39;espressione di GFP-tagged α-tubulina <ol><li> Per saggiare dinamica dei microtubuli, trasdurre le cellule con GFP-tagged α-tubulina con il controllo di trasduzione GFP commerciale (ad esempio, BacMam) secondo il protocollo del produttore. </li><li> Cultura le cellule nel pozzo per 36 h in un incubatore a 37 ° C coltura cellulare per consentire comadesione completo e il 60% di confluenza. </li><li> Calcolare il volume appropriato di GFP-tagged α-tubulina aggiungere a ciascun pozzetto, secondo la seguente equazione: <img alt="Equazione" src="/files/ftp_upload/55027/55027eq1.jpg" /> Il numero di cellule è il numero totale stimato di cellule nel pozzo al momento dell&#39;etichettatura e PPC desiderato è il numero di particelle per cella. NOTA: Semina le cellule MCF-7 va raddoppiato il numero di celle da 10 5 2 x 10 5 con un PPC desiderata del 20, il volume necessario per ogni vetrino è quindi di 40 microlitri. Per le cellule differenti, il volume calcolato sulla base della formula di cui sopra non può essere la migliore. Il volume esatto deve essere regolata in base al livello di espressione reale di GFP-tubulina. </li><li> Mescolare le alfa-tubulina più volte GFP-etichettato per inversione per garantire una soluzione omogenea. Non farlo vortice. NOTA: Per il controllo, la (controllo) reagente Null manca di qualsiasi genetica dei mammiferielementi e possono essere usati per aiutare a determinare i potenziali effetti baculovirus-mediata e fluorescenza di fondo. Un controllo di trasduzione non mirati GFP può anche essere utilizzato, che si illuminerà l&#39;intera cella. </li><li> Sostituire il terreno di coltura con terreno fresco. Aggiungere 2 ml del nuovo mezzo e 40 ml di GFP-tagged α-tubulina in ciascun pozzetto. </li><li> Agitare delicatamente la piastra per permettere la completa miscelazione di GFP-tagged α-tubulina con il terreno di coltura. Riportare la piastra alla cultura incubatore e incubare per 24 ore per permettere l&#39;espressione di GFP-tubulina. </li></ol></li><li> Trattare le cellule con docetaxel NOTA: Questo esperimento è quello di testare l&#39;effetto di docetaxel in microtubuli instabilità dinamica. Così, le cellule vengono trattate con la concentrazione desiderata di docetaxel. <ol><li> Preparare un mezzo composto 90% di DMEM e il 10% di FBS e completato con amminoacidi non essenziali. Utilizzare DMEM senza rosso fenolo perché rosso fenolo può interferire con la fluorescence di GFP-tubulina. Aggiungere la concentrazione desiderata di docetaxel (da 10 nM a 10 pM). La soluzione madre di docetaxel è di 10 mm di dimetilsolfossido (DMSO). </li><li> Pre-riscaldare il mezzo docetaxel a 37 ° C in incubatore di coltura tissutale. </li><li> Sostituire il terreno di coltura normale con il mezzo contenente docetaxel e incubare per 30 minuti nel tessuto culturale incubatore. Ci dovrebbe essere almeno 3 lamelle per pozzetto per ciascuna concentrazione docetaxel. </li></ol></li></ol> 2. l&#39;imaging dal vivo per esaminare microtubuli instabilità dinamica <ol><li> Impostazione del sistema <ol><li> Eseguire esperimenti in una camera mantenuta a 37 ° C e 5% CO 2. Acquisire immagini di fluorescenza dal lasso di tempo di un sistema microscopico dotato di 60X, 1.42 olio NA lente obiettivo su un microscopio a fluorescenza invertito e con una telecamera CCD. </li><li> Impostare le cellule per l&#39;imaging dal vivo. Pre-caldo sia il titolare del campione eil mezzo a 37 ° C. Scegliere un vetrino da esaminare. Montare il vetrino di interesse su un porta-campioni e incubare con 1 ml del mezzo contenente docetaxel preparato al punto 1.3.1. </li></ol></li><li> Immagini dal vivo di microtubuli instabilità dinamica <ol><li> Identificare le celle che verranno utilizzati per l&#39;imaging. Essi dovrebbero essere piatta, hanno una elevata intensità di fluorescenza di espresso GFP-tubulina, e hanno strutture microtubuli chiare. </li><li> Scegliere una cella dal gruppo di cellule identificate dal punto 2.2.1 di esaminare prima. Focus sulla superficie periferica della cella identificata. Impostare il programma per il tempo di esposizione e la frequenza di acquisizione delle immagini. Mentre il tempo di esposizione è di solito di 0,5 s, le immagini saranno acquisite ogni due s. </li><li> Lasciare un ulteriore 20 minuti per i passi da 1,33 a 2.2.3. Così, esattamente 50 minuti dopo l&#39;aggiunta del docetaxel, come descritto al punto 1.3.3, avviare l&#39;imaging (questo è per la standardizzazione). </li><li> Registrare il microtubulo dynamics per 2 minuti, scattare una foto ogni 2 s. In totale, acquisire 60 immagini per la cella identificata. </li><li> Passare alla successiva cella identificata e ripetere i passaggi 2.2.2 a 2.2.4. Ripetere la procedura fino a 5 celle sono state ripreso. È indispensabile farlo prima del tempo raggiunge 70 min dopo l&#39;aggiunta del docetaxel, come descritto al punto 1.3.3. </li><li> Per ogni condizione di trattamento, ripetere i passaggi da 2.2.1 a 2.2.5 per 3 diverse lamelle. In totale, immagine 15 celle, che è abbastanza dati per misurare microtubuli instabilità dinamica. </li></ol></li></ol> 3. Elaborazione delle immagini e analisi dei dati <ol><li> Deconvoluzione di immagini <ol><li> Deconvolve le immagini acquisite per ottenere una qualità elevata. Aprire il programma deconvoluzione equipaggiato con il sistema del microscopio. </li><li> Fai clic su &quot;Processo&quot; e selezionare &quot;Deconvolve.&quot; Trascinate il file immagine nella finestra &quot;Input&quot; e fare clic su &quot;Do.&quot; Il file di immagine verrà deconvolved, ed ilfile di deconvolved verrà salvato come un nuovo file automaticamente. </li></ol></li><li> Generazione di video <ol><li> Fare clic sul file immagine deconvolved da aprire con il software attrezzato. </li><li> Fai clic su &quot;File&quot; e selezionare &quot;Salva come film&quot;. Selezionare il &quot;formato di un film&quot; come &quot;AV&quot; e lo &quot;stile di animazione&quot; come &quot;Forward&quot;. Impostare la &quot;qualità di compressione&quot; a &quot;100%&quot; e il &quot;frame rate&quot; a &quot;5 / secondo.&quot; </li><li> Selezionare la casella di &quot;animare attraverso il tempo&quot; e fare clic su &quot;Do it&quot;. Il video verrà generato. </li></ol></li><li> Misura di microtubuli accorciamento e tasso di crescita <ol><li> Identificare i microtubuli che verranno utilizzati per la misurazione. Per ogni cella, solo pochi microtubuli potranno avere il loro accorciamento o di crescita seguiti chiaro e completo. </li><li> Esaminate ogni frame del video per identificare il fotogramma che mostra la lunghezza iniziale del microtubulo unND il telaio con il microtubulo più estesa o ridotta. Misurare la variazione di lunghezza tra i due microtubuli e il tempo trascorso tra i due telai. </li><li> Calcolare il tasso di crescita o accorciamento dividendo la variazione di lunghezza per tempo (l&#39;unità è micron / s). Per ogni condizione di trattamento, seguire almeno 10 cellule e misurare almeno 20 microtubuli. </li><li> Calcolare la media e l&#39;errore standard per ogni condizione di trattamento. Analizzare la differenza statistica tra le condizioni utilizzando il test di Student. Riportare i dati in una tabella e / o in un grafico. </li></ol></li></ol>

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Ci sono due metodi principali per misurare microtubuli instabilità dinamica: in vitro e in vivo. Nel metodo in vitro, tubulina purificato viene utilizzato per misurare instabilità dinamica dei microtubuli con time-lapse microscopia a interferenza differenziale contrasto informatica avanzata. Nel metodo in vivo, microiniettato tubulina fluorescenti, o espresso GFP-tubulina, è incorporato in microtubuli. La dinamica (crescita e accorciamento) dei microtubuli vengono quindi registrati...

<html> La principale causa di mortalità per cancro al seno è attraverso le metastasi 1, 2. Taxani, come docetaxel e paclitaxel, sono attualmente utilizzati come prima linea di regimi nel trattamento del carcinoma mammario metastatico 2, 3, 4, 5, 6. Fanno parte di un gruppo di agenti microtubuli-targeting (MTA) che interrompono la dinamica dei microtubuli. Tuttavia, una delle più grandi sfide di utilizzare taxani nella terapia curativa è lo sviluppo della resistenza taxani nelle cellule tumorali, che porta alla malattia recidiva 7. La resistenza ai farmaci rappresenta oltre il 90% di tutti i decessi tra i pazienti con carcinoma mammario metastatico 7. I microtubuli sono formati dalla polimerizzazione di eterodimeri alfa- e beta-tubulinaclass = &quot;xref&quot;&gt; 8, 9. La regolazione precisa della dinamica dei microtubuli è importante per molte funzioni cellulari, tra cui la polarizzazione delle cellule, progressione del ciclo cellulare, trasporto intracellulare, e segnalazione cellulare. Disregolazione dei microtubuli e le loro dinamiche interromperà la funzione delle cellule e causare la morte delle cellule 10, 11. A seconda di come essi causano questa disregolazione, farmaci MTA possono essere classificati come agenti dei microtubuli di stabilizzazione (ad esempio, taxani) o agenti microtubuli destabalizing (cioè, alcaloidi della vinca o colchicina loco agenti leganti) 20. Nonostante i loro effetti opposti sulla massa microtubuli, ad una dose sufficiente, entrambe le classi possono uccidere le cellule tumorali attraverso i loro effetti sulla dinamica dei microtubuli 21. Taxani funzionano principalmente dalla stabilizzazione del microtubulo del mandrino 12, che porta adisallineamento cromosomica. La successiva attivazione perpetua del punto di controllo di assemblaggio del mandrino (SAC) arresta la cella in mitosi. Mitotico arresto prolungato provoca quindi l&#39;apoptosi 13, 14. Taxano interagisce con microtubuli attraverso il sito di legame taxano on β-tubulina 8, 15, che è presente solo in tubulina assemblato 16. Meccanismi multipli per resistenza taxani sono stati proposti 9, 17. Questi meccanismi comprendono sia la resistenza ai farmaci in generale a causa della sovraespressione di proteine droga efflusso e specifici taxano resistenza 5, 9, 18, 19. Ad esempio, le cellule tumorali taxani resistenti potrebbero aver alterato espressione e la funzione di alcuni β-tubulin isotipi 5, 9, 19, 20, 21, 22, 23. Utilizzando un metodo in vivo per misurare microtubuli instabilità dinamica, dimostriamo che, rispetto ai non-resistenti, parentali cellule MCF-7 CC 17, la dinamica dei microtubuli di cellule MCF-7 TXT docetaxel-resistenti sono insensibili al trattamento con docetaxel. Per capire meglio la funzione di MTA e l&#39;esatto meccanismo di taxano-resistenza in cellule cancerose, è essenziale misurare dinamica dei microtubuli. Qui riportiamo un metodo in vivo di farlo. Utilizzando immagini dal vivo, in combinazione con l&#39;espressione di tubulina GFP-tagged nelle cellule, siamo in grado di misurare la dinamica dei microtubuli di MCF-7 TXT e le cellule CC MCF-7 con e wisarie, senza trattamento con docetaxel. I risultati possono aiutarci a progettare farmaci più efficaci che possono superare la resistenza taxano.

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			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Life Technologies, Invitrogen</td>
			<td>11140-050</td>
			<td></td>
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			<td>FBS</td>
			<td>Gibco, Invitrogen</td>
			<td>12483</td>
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			<td>Anti-Anti (100x)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15240-062</td>
			<td></td>
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			<td>docetaxel</td>
			<td>Sigma-Aldrich</td>
			<td>01885-5mg-F</td>
			<td></td>
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			<td>DMEM phenol red-free</td>
			<td>Gibco, Invitrogen</td>
			<td>21063</td>
			<td></td>
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			<td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td>
			<td>ThermoFisher Scientific</td>
			<td>C10613</td>
			<td>Key reagent for expressing GFP tubulin in cells</td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP</td>
			<td>ThermoFisher Scientific</td>
			<td>B10383</td>
			<td>Control</td>
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		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich+B9:AA9</td>
			<td>472301</td>
			<td>for dissoving decetaxel</td>
		</tr>
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			<td>22-mm glass coveslip</td>
			<td>Fisher Scientifics</td>
			<td>12-545-101</td>
			<td></td>
		</tr>
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			<td>6-well culture plate</td>
			<td>Greiner Bio-One International</td>
			<td>6 Well Celi Culture Plate</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Microscopy Imaging Systems</td>
			<td>GE Health</td>
			<td></td>
			<td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td>
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			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemacytometer Set, Hausser Scientific</td>
			<td>Hausser Scientific, Distributed by VWR</td>
			<td>Supplier No.: 1492 VWR No.:15170-172</td>
			<td></td>
		</tr>
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live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

Utilizzando il protocollo presentato qui, abbiamo studiato gli effetti del docetaxel sulla dinamica dei microtubuli del normale (MCF-7 CC) e docetaxel-resistente (MCF-7 TXT), le cellule del cancro al seno. Due serie di immagini mostrano gli effetti di docetaxel (0,5 mM) sulla crescita microtubuli e accorciamento in MCF-7 CC e MCF-7 TXT cellule (Figura 1A). Abbiamo in...

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

Immagini dal vivo per studiare Microtubulo instabilità dinamica nei tumori al seno taxani resistente

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

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7.5K Views.  University of Alberta. In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. 

Video: Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

Utilizzando Genomica Funzionale screening per identificare gli obiettivi farmaco potenzialmente romanzo in Cancer Cell Culture Spheroid

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

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A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Un modello di iniezione vena porta del fegato per studiare metastasi del cancro al seno

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

Live-imaging della migrazione delle cellule epiteliali della mammella dopo lo svuotamento transitorio di TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

A lungo termine Live cell Imaging per valutare il destino delle cellule in risposta a Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Analisi Live Cell Imaging Studio Glioblastoma cerebrale tumore cellule staminali migrazione e invasione

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Studiare il cancro al seno triplo negativo utilizzando il modello di cancro al seno ortotopico

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Utilizzo del modello di membrana coroallantoica di pollo in vivo per studiare i tumori ginecologici e urologici

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Utilizzo dell'analisi delle immagini basata su computer per migliorare la quantificazione della metastasi polmonare nel modello di cancro al seno 4T1

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Modellazione del cancro al seno nel tessuto mammario umano utilizzando un sistema microfisiologico

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Un modello murino di ommaya xenotrapianto per studiare la terapia mirata diretta della malattia leptomeningea

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...