Siamo grati a Ken Iwata (OSI Pharmaceuticals, New York, USA) per i reagenti e Fred Miller (Barbara Ann Intitute Karmanos Cancer, Detroit, USA) per le linee cellulari.

1. Preparazione della soluzione METHOCEL (500 ml) <ol><li> Autoclave 6 g di metilcellulosa in una bottiglia da 500 ml contenente un agitatore magnetico. </li><li> Sciogliere la metilcellulosa in 250 ml di DMEM/F12 preriscaldato senza siero (60 ° C) per 20 min. </li><li> Aggiungere 250 ml di DMEM/F12 (RT) con siero di cavallo 10%. Mix notte (4 ° C). </li><li> Chiara la soluzione per centrifugazione (5000g, 2 ore, 4 ° C). </li><li> Prendere la soluzione limpida altamente viscosi a un nuovo tubo (circa il 90-95% della soluzione di riserva). </li><li> Conservare a 4 ° C fino all&#39;utilizzo. </li></ol> 2. Cultura sferoide <ol><li> Preparare una soluzione al 20% METHOCEL (10 ml METHOCEL e 40 ml di terreno di coltura). </li><li> Lavare le cellule due volte con PBS, aggiungere lo 0,1% tripsina e cellule incubare a 37 ° C </li><li> Risospendere le cellule in terreno di crescita e contare le cellule. </li><li> Preparare una sospensione di 10 000 cellule per ml nel 20% METHOCEL. </li><li> Aggiungere la sospensione cellulare ai 96 fondo anche l&#39;spiastre uspension (100 l per pozzetto). </li><li> Il giorno dopo, controlla la formazione sferoide nei piatti sospensione. Sferoidi sarà pronto per l&#39;uso dopo 1-3 giorni. </li></ol> 3. Preparazione del collagene <ol><li> Preparare sul ghiaccio una soluzione di 8 collagene ml, 1 ml di PBS 10x, 1 ml di NaOH 0,1 N e 3 gocce di 0,1 N HCl. Controlla se il pH è di 7,4 individuando alcune delle soluzione sulle strisce indicatore di pH. Regolare il pH se fuori portata. </li><li> Aggiungere 50 ml di soluzione di collagene neutralizzata nei pozzetti di una piastra a 96 pozzetti </li><li> Incubare a 37 ° C fino a quando il collagene è solidificata (90 min). </li><li> Preparare METHOCEL-collagene-ligando soluzioni su ghiaccio: per ogni ligando pipetta 1 parte di soluzione di collagene. Aggiungere 20 ng per ml di collagene TGF-β. Dopo diluizione con METHOCEL e diffusione sui diversi strati, la concentrazione finale di TGF-β sarà di 5 ng / ml Mescolare vortexando. Aggiungere 1 parte di METHOCEL-soluzione. Mescolare nel vortex. </li></ol> 4. Sferoide incorporareding <ol><li> Inserire una punta di 200 microlitri, che ha circa 5 mm dalla punta tagliata, in una pipetta 200 ul. Aspirare uno sferoide con la pipetta e dispensare con cura il mezzo in un piatto vuoto. Guarda se la sferoide rimane all&#39;interno della punta. Il sferoide è visibile come un punto bianco. Senza rilasciare lo stantuffo, aspirare 100 l collagene METHOCEL miscela e dispensare lo sferoide nella miscela di collagene in un collagene rivestito 96 pozzetti. Fate questo per 12 sferoidi per ogni condizione. </li><li> Lasciate solidificare il collagene almeno 30 minuti a 37 ° C </li><li> Rimuovere le bolle d&#39;aria con una punta di 200 l. </li><li> Aggiungere 50 ml di media con 1,6% di siero e gli inibitori sciolto in DMSO. Per SB-431542 aggiungere 4 ml di soluzione madre (10 mM) a 1 ml di terreno. Dopo la diffusione, la concentrazione finale della inibitore sarà di 10 micron. TGF-β e gli inibitori rimarrà il corso dell&#39;esperimento. Scatta foto di sferoidi al microscopio con ingrandimento 40x. </li><li> Dopo 48h di stimolazione con TGF-β e / o inibitori, scattare foto degli sferoidi al microscopio con ingrandimento 40x. </li><li> Misurare l&#39;area del sferoidi invasori dopo 2 giorni e sottrarre la zona di partenza. Misurazione della superficie di uno sferoide: Aprire l&#39;immagine dal sferoide in Adobe Photoshop Extended (figura 2A) e selezionare lo strumento di selezione rapida (figura 2B). Il puntatore del mouse si trasforma in un cerchio con un &#39;plus&#39; (+) segno (figura 2C). Mentre si trascina il cursore sopra la sferoide, Photoshop riconoscerà i confini della sferoide. Se una zona diversa da sferoide è selezionata, questa regione può essere esclusa dalla selezione premendo il tasto Alt o utilizzando lo strumento di selezione negativa, che è indicata da un segno (-) nella barra degli strumenti. Il cursore si trasforma in un cerchio con un &#39;meno&#39; (-) firmare e trascinando il cursore sopra l&#39;area erroneamente selezionata, questa zona viene rimosso dalla selezione (figura 2D). Se necessario, più regioni può essere selezionato premendo il Shift pulsante. Per lavorare con maggiore precisione, le dimensioni del puntatore del mouse può essere regolata cambiando il diametro del pennello nella barra degli strumenti (2E figura). Quando la sferoide tutto è selezionata, l&#39;area della selezione viene calcolata tramite ANALISI SU MISURA nella barra dei menu o premendo Ctrl + Maiusc + M (figura 2F). La finestra di registrazione misura apparirà mostrando il nome del file e la zona della selezione in pixel, così come altri dati (figura 2G). Se selezioni multiple sono misurati, la prima riga indica la somma di tutte le aree. Le misure delle scelte individuali sono presentati nelle linee di basso. I dati di tutte le misurazioni possono essere esportati in un file di testo e aperto in Excel. </li></ol> 5. Rappresentante dei risultati: Un esempio di saggio sferoide con MCF10A1 (M1), le cellule epiteliali mammarie normali, H-RAS trasformato MCF10AneoT (M2), le cellule e M2-derivati ​​MCF10CA1a (M4) è dato in Figura 3. M1 cellule hanno mostrato solo debole senza invasionestimolazione, ma invaso significativamente migliore dopo la stimolazione da TGF-β. Al contrario, la RAS-trasformata M1-M2 derivati ​​già invaso in modo efficiente senza aggiunta di stimoli, e questo è stato ulteriormente aumentato 4-5 volte su TGF-β trattamento. M4 cellule hanno mostrato la più forte invasione, con o senza aggiunta di TGF-β. Successivamente, abbiamo valutato se ci potrebbe interferire con TGF-β-indotta invasione in questo modello sferoide utilizzando inibitori chimici. A questo scopo abbiamo utilizzato SB-431542, un analogo ATP e selettivo inibitore dell&#39;attività della chinasi TβRI, così come activina recettore di tipo IB (ActRIB) e activina receptor-like kinase (ALK) 7 12. Come previsto, SB-431542 potentemente inibito TGF-β-indotta invasione di M1, M2 e M4 cellule (Fig. 4), indicando che TGF-β-indotta invasione è TβRI-chinasi dipendente. Inoltre, l&#39;invasione basale della sferoidi è stata fortemente inibita, il che suggerisce che l&#39;invasione della base è anche deindipendenti sul TGF-β. <img alt="Figura 1" src="/files/ftp_upload/3337/3337fig1.jpg" /> Figura 1. Diagramma di flusso del test 3D sferoide. In primo luogo, le cellule sono placcati in condizioni non aderente a forma di u piastre sospensioni. Cellule aggregrate in sferoidi multcellular e può essere incorporato in una matrice di collagene. In questa matrice di collagene, sono in grado di invadere. <img alt="Figura 2" src="/files/ftp_upload/3337/3337fig2.jpg" /> Figura 2. Quantificazione dell&#39;area del sferoidi utilizzando Adobe Photoshop Extended. (A) Il quadro della sferoide è aperto in Adobe Photoshop allargata del (B) Selezione lo strumento Selezione rapida (C) Trascinando il cursore sopra la sferoide per selezionare l&#39;area dello sferoide (D) La rimozione di una zona di wongly incluso usando l&#39; negativo veloce strumento Selezione (E) Regolazione della dimensione del pennello dello strumento di selezione rapida per selezionare con maggiore precisione l&#39;area (F) Selezione delmisurazione comando da registrare (G) Esempio di un record di misura. <img alt="Figura 3" src="/files/ftp_upload/3337/3337fig3.jpg" /> Figura 3. TGF-β-indotta invasione di sferoidi spontaneamente immortalata MCF10A1 (M1) (A), RAS-trasformato MCF10AneoT (M2 (B) e le sue metastasi derivate MCF10CA1a (M4) (C). M1, M2 e M4 sferoidi incorporato in collagene sono stati trattati con 5ng/ml di TGF-β per 48 ore, come indicato. AC immagini rappresentative tratte al giorno di incasso e due giorni dopo. invasione D relativo è stato quantificato come differenza zona giorno 2 meno giorno 0. I risultati sono espressi come media ± sd Adattato da 13. <img alt="Figura 4a" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="Figura 4b" src="/files/ftp_upload/3337/3337fig4b.jpg" /> Invasione sferoide Figura 4. Basale e TGF-β-indotta è inibita by SB-431542. Sferoidi M1, M2 e M4 sono stati incorporati nel collagene e trattati con 5ng/ml TGF-β e / o 10 mM SB-431542 come indicato. Immagini rappresentative tratte dopo 2 giorni (A). Invasione parente è stato quantificato come differenza zona giorno 2 meno giorno 0 (B). I risultati sono espressi come media ± sd Adattato da 13.

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Nessun conflitto di interesse.

Abbiamo stabilito un modello di sferoide in cui MCF10A1 cellule e dei suoi derivati ​​maligne invadono in una matrice di collagene in un TGF-β-dipendente. In primo luogo, sferoidi si formano in 96-pozzi di piastre di fondo sospensione rotonda alla presenza di metilcellulosa. Naturalmente, anche a forma di U piatti rivestiti di poli-HEMA può essere utilizzato per questo scopo. Metilcellulosa è assolutamente necessaria in questa fase, poiché le cellule moriranno in assenza di metilcellulosa. TGF-β viene aggiunto ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Metilcellulosa </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Avanzate Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> Indicatore di pH striscie </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96-oltre sospensione Piastre </td><td> Greiner Bio-one </td><td> 650 185 </td><td></td></tr><tr><td> 96-TC e l&#39;adesione Piastre </td><td> Greiner Bio-one </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF-β ha opposti ruoli nella progressione del cancro al seno, agendo come un soppressore del tumore nella fase iniziale, ma stimolando l&#39;invasione e metastasi in seguito 1,2 stadio. Inoltre, TGF-β è spesso sovra-espresso nel cancro al seno e la sua espressione correla con prognosi infausta e metastasi 3,4. I meccanismi attraverso i quali TGF-β induce invasione non sono ben compresi. TGF-β suscita le sue risposte via cellulare di tipo II del TGF-β (TβRII) e di tipo I (TβRI) recettori. Su TGF-β-indotta formazione eteromerici complesso, TβRII fosforila il TβRI. Il TβRI attivato avvii un percorso di segnalazione intracellulare canonica da Smads fosforilazione del recettore (R-Smads), cioè Smad2 e Smad3. Questi attivato R-Smads formare complessi con eteromerici Smad4, che si accumulano nel nucleo e regolano la trascrizione dei geni bersaglio 5. Oltre al già described percorso Smad, i risultati del recettore attivazione attivazione di numerose altre vie di segnalazione non Smad, per esempio mitogeno proteina chinasi attivata (MAPK) percorsi di 6. Per studiare il ruolo del TGF-β in diversi stadi di cancro al seno, abbiamo fatto uso del sistema a celle MCF10A. Questo sistema è costituito da immortalata spontaneamente MCF10A1 (M1), le cellule epiteliali del seno 7, l&#39;H-RAS trasformato M1-derivato MCF10AneoT (M2), che produce lesioni precancerose nei topi 8, e l&#39;M2-derivato MCF10CA1a (M4), istituito dalla xenotrapianti M2 e forme ad alta carcinomi di grado con la capacità di metastatizzare al polmone 9. Questa serie MCF10A offre la possibilità di studiare le risposte delle cellule con diversi gradi di malignità che non sono viziate da un background genetico diverso. Per l&#39;analisi di TGF-β-indotta invasione, abbiamo generato homotypic MCF10A sferoide embe colture cellularidded in una matrice di collagene 3D in vitro (Fig. 1). Tali modelli sono molto simili tumori umani in vivo mediante l&#39;istituzione di un gradiente di ossigeno e nutrienti, con conseguente cellule attive e invasivi sulle cellule all&#39;esterno e quiescenti o addirittura necrotico nella parte interna della sferoide 10. Sferoide test basato hanno anche dimostrato di ricapitolare meglio la resistenza ai farmaci di colture monostrato 11. Questo modello MCF10 sistema 3D ci ha permesso di studiare l&#39;impatto di TGF-β segnalazione sulle proprietà invasiva delle cellule del seno in diverse fasi di malignità.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-ÃŽÂ²-induced Invasion at JoVE.com

Spheroid Assay to Measure TGF-&#946;-induced Invasion

Un test per misurare quantitativamente fattore di crescita trasformante (TGF)-β-indotta invasione in 3-dimensionale gel collagene è descritto. Questo saggio si avvale della MCF10A serie di linee cellulari, che rappresentano diversi stadi di sviluppo del cancro al seno. Questo metodo può essere adottato per essere utilizzato con altre linee cellulari e può essere utilizzato per indagare altri potenziali attivatori o inibitori di invasione.

Saggio sferoide Misura TGF-β-indotta Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

spheroid-assay-to-measure-tgf-induced-invasion

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Medicine

21.8K Views.  Leiden University Medical Centre. Un test per misurare quantitativamente fattore di crescita trasformante (TGF)-β-indotta invasione in 3-dimensionale gel collagene è descritto. Questo saggio si avvale della MCF10A serie di linee cellulari, che rappresentano diversi stadi di sviluppo del cancro al seno. Questo metodo può essere adottato per essere utilizzato con altre linee cellulari e può essere utilizzato per indagare altri potenziali attivatori o inibitori di invasione.

Video: Spheroid Assay to Measure TGF-β-induced Invasion

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

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

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

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

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

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Medicina

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modellazione e imaging 3-Dimensional Invasion cella Collettiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

Metodo per misurare il tono muscolare di assiale e prossimale

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Accuratezza nella Dental Medicine, Un nuovo modo di misurare veridicità e precisione

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

A Modified<em&gt; In vitro</em&gt; Invasion test per determinare il ruolo potenziale di ormoni, citochine e / o fattori di crescita nella mediazione Cancer Invasion cellulare

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Valutazione del cancro ovarico Spheroid attaccamento e l&#39;invasione delle cellule mesoteliali in tempo reale

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetria intrauterina misurare mouse contrattile Pressione<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

Invasione Chick Cuore Saggio per il test l&#39;invasività delle cellule tumorali e l&#39;attività di composti potenzialmente anti-invasive

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Un Cancer Cell Saggio sferoide valutare Invasion in un Ambiente 3D

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...