Name: live imaging to study microtubule dynamic instability in taxane-resistant breast cancers
Uploaded: 2017-02-20T13:00:00.000+00:00
Duration: 6 min 2 s
Description: Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

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.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;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></tr><tr><td>FBS</td><td>Gibco, Invitrogen</td><td>12483</td><td></td></tr><tr><td>Anti-Anti (100x)</td><td>Life Technologies, Invitrogen</td><td>15240-062</td><td></td></tr><tr><td>docetaxel</td><td>Sigma-Aldrich</td><td>01885-5mg-F</td><td></td></tr><tr><td>DMEM phenol red-free</td><td>Gibco, Invitrogen</td><td>21063</td><td></td></tr><tr><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></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich+B9:AA9</td><td>472301</td><td>for dissoving decetaxel</td></tr><tr><td>22-mm glass coveslip</td><td>Fisher Scientifics</td><td>12-545-101</td><td></td></tr><tr><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></tr><tr><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></tbody></table>

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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Cell Biology

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Cell Division

The Cytoskeleton II: Microtubules and Intermediate Filaments

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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

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 ...

Ricerca

Ricerca sul cancro

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Monitoraggio metastasi tumorali e lesioni osteolitiche con bioluminescenza e Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Medicina

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

In Imaging bioluminescenza in vivo di Dinamica Tumore ipossia di metastasi cerebrali cancro al seno in un modello murino

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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 Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Vivo Imaging di risposte droga nel microambiente tumorale in modelli murini di cancro al seno

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse imaging di primaria preneoplastiche cellule epiteliali mammarie Derivato da modelli murini geneticamente modificati di cancro al seno

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

In tempo reale Imaging di cellule mieloidi Dynamics in<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinale tumori da Spinning Disk Microscopia confocale

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Immunologia e infezioni

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Visualizzazione simultanea della dinamica di microtubuli reticolati e singoli in vitro mediante microscopia TIRF

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Biochimica

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Attraverso lo specchio: Time-lapse Microscopia e longitudinale monitoraggio di singole cellule allo Studio Therapeutics anti-cancro

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Esteso Time-lapse Imaging intravitale del Real-time multicellulare Dynamics nel microambiente tumorale

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...