Name: three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation
Uploaded: 2016-08-18T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

NOTA: Non etica considerazione dal momento che le cellule tumorali umane e animali sono stati acquistati o gentilmente forniti da noi. 1. Fare collagene spine (Pseudo-primaria del tumore) <ol><li> Preparare una dispersione di collagene. Collagene di tipo I da tendini coda di topo (RTT) possono essere estratti sia e sterilizzati come riportato in precedenza 17, o acquistati. Disperse collagene liofilizzato RTT (3,25-3,50 mg / ml in 0,02 N acido acetico) utilizzando un miscelatore (impostazione alta velocità; cinque 2 min funzionamenti) per una miscelazione uniforme. </li><li> Harvest (tripsina-EDTA, di solito) e utilizzare esclusione trypan blu per il conteggio delle cellule vitali con un emocitometro. Regolare la densità cellulare desiderato (5 x 10 4 cellule per spina). </li><li> Preparare tutte le soluzioni (NaOH, siero fetale bovino, DMEM 5x, NaHCO 3) separatamente (Tabella 1) in condizioni sterili e mantenere refrigerata in ghiaccio. Nota: L&#39;ordine di aggiunta dei vari soluzioni è importante per evitare shock osmotici o acidi nelle cellule. </li><li> Eseguire dispersione cella (1,25 x 10 6 cellule) nella soluzione di collagene finale (5 ml) più rapidamente possibile. Mescolare bene (pipettando su e giù) evitando bolle d&#39;aria, e quindi distribuire rapidamente 200 microlitri della soluzione pronta all&#39;uso in ciascun pozzetto di una piastra a 96 pozzetti. colpire delicatamente la piastra multi-bene sulla superficie della cappa di coltura cellulare di lavoro per rimuovere le bolle d&#39;aria e diffondere uniformemente la soluzione all&#39;interno dei pozzetti. </li><li> Dopo aver riempito fino tutti i pozzetti (questo passaggio richiede circa 15-20 minuti per ogni piastra a 96 pozzetti), memorizzarlo in incubatrice. </li><li> Incubare la piastra a 37 ° C da 2 ore a notte. Il collagene gelificazione (cioè, fibrillogenesi) si verifica entro 30 min. Aggiungere terreno di coltura (100 ul / pozzetto) alla cultura per eseguire una notte di incubazione. </li></ol> 2. Primo strato di fibrina gel <ol><li> Fibrinogeno preparazione della soluzione. NOTA: Lo stesso lotto di gel di fibrina dovrebbe idealmente essere utilizzato per Resul più riproducibilets, formazione di gel di fibrina come può variare tra i diversi lotti di fibrinogeno liofilizzato commerciale. <ol><li> Usare sempre una soluzione di fibrinogeno preparati al momento. Portare il fibrinogeno liofilizzato a temperatura ambiente prima di aprire il flacone per evitare la formazione di cristalli di idrati. </li><li> Progressivamente sciogliere il fibrinogeno in pre-riscaldato (37 ° C) soluzione salina bilanciata di Hank (HBSS) con Ca 2+ / Mg 2+ ad una concentrazione di lavoro di 3 mg / ml (non preparare un eccesso del 15% del volume finale minima necessaria : ad esempio, 17.25 mg a 5,75 ml di soluzione 5 ml). <ol><li> Aggiungere pre-riscaldato a gocce HBSS in un primo momento per solubilizzare frammenti di fibrinogeno. Abbattere i frammenti più grandi con una spatola nel bicchiere. Agitare il bicchiere di volta in volta a facilitare la miscelazione. Non usare una piastra di agitatore durante la procedura. Sciogliere la polvere rimanente pipettando sospensione su e giù. </li></ol></li><li> Conservare la soluzione di fibrinogeno tiepida mentre sterilizing la soluzione passa attraverso un filtro di 0,22 micron. Nota: se il HBSS non è abbastanza caldo o il fibrinogeno non completamente dissolto, la soluzione può intasare il filtro. Se del caso, sostituire il filtro una o due volte, che possono ridurre la concentrazione di fibrinogeno, e la rigidità quindi coagulo di fibrina. </li><li> Sospendere le cellule di interesse (ad esempio, cellule endoteliali) in soluzione di fibrinogeno pronta per l&#39;uso durante la regolazione il volume finale, quale procedura alternativa. </li></ol></li><li> Preparare le soluzioni di trombina. <ol><li> Preparare una soluzione madre in DDH 2 O (50 NIH unità / ml), quindi sterilizzarlo con un filtro 0,22 micron. </li><li> Utilizzare un rapporto / trombina fibrinogeno ≥1: 0,0075 (v / v) per generare il gel di fibrina. </li></ol></li><li> Generazione del gel di fibrina. <ol><li> Tenere il fibrinogeno e trombina sterili soluzioni stock su ghiaccio durante tutte le fasi successive. I gel di fibrina possono formare in piastre da 24 pozzetti. </li><li> Prontamente sovrapposizione °e la superficie di ciascun pozzetto con la soluzione di fibrinogeno (200 pl / pozzetto) evitando la formazione di bolle d&#39;aria. Processo 6 pozzetti alla volta. </li><li> Una volta che la soluzione di fibrinogeno copre completamente la superficie dei pozzetti, inclinare il piatto in un angolo di 45 ° e aggiungere 1,5 ml di soluzione di trombina al primo pozzo facendo cadere la trombina al centro del pozzo, e poi ruotare delicatamente la piastra orizzontale per 1-2 sec. <ol><li> Lasciare la piastra in posizione stabile sotto cappa a flusso laminare (5-10 min) fino a quando il processo di gelificazione / coagulazione ha completato (NB: il processo di polimerizzazione non deve essere disturbato, ad esempio, trasportando la piastra alla incubatore). </li></ol></li><li> Una volta che i primi sei pozzi sono polimerizzati, ripetere la stessa sequenza (cioè i 3 passaggi precedenti) per i prossimi sei pozzi fino a quando tutti i pozzetti sono stati elaborati. </li></ol></li></ol> 3. secondo strato di fibrina gel e sandwich collagene Plug <ol><li> OpzioneA: (Utilizzo del plug collagene immediatamente). <ol><li> Assicurarsi che il primo strato di gel di fibrina è polimerizzato in tutti i pozzetti con delicatezza, inclinando la piastra. Posizionare la piastra a 96 pozzetti contenente lato tappi di gel di collagene fianco con piastra da 24 pozzetti (contenente gel di fibrina) per facilitare il trasferimento dei tappi di collagene. </li><li> Aggiungere una goccia di HBSS in ciascun pozzetto della piastra contenente i tappi di collagene. </li><li> Rimuovere ciascun tappo collagene dal pozzo con un ago sottile montato su una siringa (usato come una maniglia) o utilizzando un micro-cucchiaio (vedi video). Trasferire ogni spina collagene sul primo strato di gel di fibrina utilizzando uno o due micro-cucchiai, facendo in modo che il tappo di collagene sia ben centrato nel bene e che la sterilità è ben mantenuto. </li><li> Sovrapporre il gel di fibrina precedentemente formato con il secondo strato di soluzione di fibrinogeno (300 ml / pozzetto) e introdurre la trombina come descritto in 2.3, mantenendo una minima 1:. Rapporto di 0,0075 e una sequenza di sei pozzetti alla volta &lt;/ Li&gt; </ol></li><li> Opzione B (rivestimento Plug collagene con un sottile strato di Growth Factor-ridotto membrana basale (GFRBM)). <ol><li> Raffreddare tutte le soluzioni preparate e gli strumenti in anticipo e tenerli a 4 ° C o su ghiaccio (ad esempio, pipette, consigli, provette) durante la movimentazione da aliquote congelate di GFRBM sono molto sensibili alla velocità di riscaldamento eccessivo durante lo scongelamento (seguire le istruzioni del produttore) . </li><li> Dopo la rimozione dai pozzetti della piastra, immergere ogni spina collagene per 2 minuti in una provetta da centrifuga da 1,5 ml su ghiaccio contenente 100 ml di una soluzione GRFBM puro. </li><li> Trasferire ciascun tappo rivestito sul primo strato di fibrina assicurando è ben centrata, come descritto in precedenza. Incubare le piastre a innesto contenente a 37 ° C per 5 minuti per consentire la GRFBM per formare un gel. Aggiungere il secondo strato di fibrina come al punto 3.1.4. </li></ol></li></ol> 4. colture cellulari condizioni medie <ol><li> Riempire ogni pozzetto con terreno di coltura (400 ml). I terreni di coltura e supplementi saranno selezionati in base alla linea cellulare e condizioni sperimentali. </li><li> Aggiungere aprotinina, un agente antifibrinolitico, al mezzo di coltura ad una concentrazione finale di 100 unità di inibitore callicreina (KIU) / ml. NOTA: Conservare le piastre in un incubatore di coltura cellulare nelle condizioni utilizzate per la linea cellulare testato. </li><li> Rifornire culture con terreno fresco ogni altro giorno o in base alla pianificazione sperimentale, e aggiungere aprotinina. Prima di aggiungere mezzo fresco, inclinare leggermente la piastra (con un angolo di 30-35 °) e inclinare il pipetta contro il lato del bene mentre accuratamente aspirazione terreno condizionato sotto costante osservazione. </li></ol>

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The authors have no disclosure.

Come importante nota tecnica, è essenziale che non gap è presente all&#39;interfaccia tra la centrale ei gel periferici. Altrimenti, si potrebbe ridurre la capacità delle cellule di migrare / invadere il gel di fibrina. Uno spazio tra il collagene e gel di fibrina possono formarsi durante il primo 24 ore di coltura se trombina non è stato opportunamente diluito. È anche possibile che la linea cellulare testato potrebbe portare il gel di collagene per contrarre durante la coltura, causando così uno spazio relativam...

Indagare le caratteristiche fondamentali e biomediche di invasione delle cellule tumorali / migrazione e la successiva creazione di metastasi è oggetto di un intenso lavoro di ricerca 1,2. Metastasi è la fase finale di cancro e la sua gestione clinica resta sfuggente. Una migliore comprensione di metastasi a livello cellulare e molecolare consentirà lo sviluppo di terapie più efficaci 3. Diverse proprietà di cellule metastatiche possono essere esplorate in vitro 4 compreso il loro stemness e potenziale per acquisire uno stato di transizione (per esempio, epithelioid-mesenchimale transizione) di migrare e invadere all&#39;interno e dal tumore primario 5. Tuttavia, la valutazione in vitro di processi / metastasi invasione è stata una sfida poiché esclude praticamente il contributo del sangue / circolazione linfatica. colture organotipiche che incorporano frammenti di tumore in gel di collagene hanno Precesornione stato utilizzato per monitorare l&#39;aggressività del cancro. Anche se la complessità dei tumori è conservata (ad esempio, la presenza di cellule non cancerose), frammenti di tumore sono esposti a limitata diffusione media, la variazione di campionamento, e ad una crescita eccessiva di cellule stromali 6. Un metodo alternativo consiste nel crescere cellule tumorali all&#39;interno dei componenti della matrice extracellulare (ECM), che imita l&#39;ambiente cellulare tridimensionale (3D). La proliferazione di linee cellulari di cancro al seno in un gel di collagene e / o una matrice membrana basale-derivato è tra gli esempi meglio caratterizzati di coltura cellulare 3D. Utilizzando specifici ambienti di coltura cellulare 3D, il gruppo disorganizzato osservata per le cellule del cancro al seno coltivate in condizioni standard può essere invertito per la formazione spontanea di acini mammaria e strutture tubolari 7-10. Inoltre, la formazione di sferoidi tumorali multicellulari derivate da cellule tumorali adenocarcinoma congregata con tecniche diverse (ad esempio, appeso gocce, sferoidi galleggiante, agar embedment) ora costituisce la coltura delle cellule 3D test più comunemente usato 11-13. Tuttavia, questo test è limitata dal ristretto insieme di linee cellulari tumorali che possono formare sferoidi e dal breve periodo a disposizione per studiare le cellule in queste condizioni. In questa tecnica visualizzata, abbiamo qui introduciamo un sofisticato test di coltura cellulare 3D in cui le cellule tumorali di interesse sono incorporati in un gel di collagene per consentire la formazione in vitro di un tumore pseudo-primario che può essere alternativamente rivestita con una matrice di membrana derivate interrato. Una volta formato, il tumore pseudo-primaria viene poi inserita in una matrice acellulare (gel di fibrina nel presente caso), che permette alle cellule tumorali di attraversare l&#39;interfaccia tra i due compartimenti matrice (vedi Figura 1). È interessante notare che le strutture di tumore-come secondarie provenienti dal tumore pseudo-primaria con le cellule tumorali aggressive appaiono nelgel di fibrina. Tale sistema di coltura 3D offre la flessibilità necessaria per indagare, ad esempio, farmaci antitumorali, espressione genica e cellula-cellula e / o interazioni cellula-ECM 14-16. <img alt="Figura 1" src="/files/ftp_upload/54322/54322fig1.jpg" /> Figura 1:.. Panoramica del metodo di sintesi schematica del metodo per generare il sistema di coltura cellulare 3D come modello per lo studio del cancro <a href="https://www.jove.com/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

<table><tbody><tr><td>Freeze-dried collagen</td><td>Sigma-Aldrich</td><td>C7661</td><td>from rat tail tendon (soluble dispersion) or home-made (see Rajan &lt;i&gt;et al&lt;/i&gt;., ref.#14)</td></tr><tr><td>Fibrinogen (freeze-dried)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F8630</td><td>Type I-S, 65 - 85% protein with &amp;ge; 75% of protein is clottable</td></tr><tr><td>Thrombin</td><td>EMD Chemicals Inc.</td><td>605157</td><td>&amp;nbsp;Gibbstown, NJ; NIH units/mg dry weight&amp;nbsp;</td></tr><tr><td>Growth factor-reduced Matrigel&amp;nbsp;</td><td>Corning</td><td>356234</td><td>Previously from BD Biosciences</td></tr><tr><td>Aprotinin</td><td>Sigma-Aldrich</td><td>A6279&amp;nbsp;</td><td>&amp;nbsp;solution at 5 - 10T IU/ml (Trypsin Inhibitor Unit)&amp;nbsp;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Micro-spoons</td><td>Fisher Scientific</td><td>2140115</td><td>Fisherbrand Handi-Hold Microspatula</td></tr><tr><td>96 well plate, round base</td><td>Sarstedt</td><td>3925500</td><td></td></tr><tr><td>24 well plate</td><td>Sarstedt</td><td>3922</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s Medium</td><td>Sigma Chemical, Co.</td><td>D5546</td><td>DMEM</td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>CAA15-701</td><td>FBS, Canadian origin.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma Chemical, Co.</td><td>T4049</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>Sigma Chemical, Co.</td><td>H8264</td><td>HBSS</td></tr></tbody></table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

colture cellulari di mammifero in monostrati è ampiamente usato per studiare i vari processi fisiologici e molecolari. Tuttavia, questo approccio per studiare le cellule in crescita genera spesso artefatti indesiderati. Pertanto, coltura cellulare in un ambiente tridimensionale (3D), spesso utilizzando componenti della matrice extracellulare, emerso come un&#39;alternativa interessante per la sua somiglianza al nativo nel tessuto vivo o organo. Abbiamo sviluppato un sistema di coltura cellulare 3D utilizzando due compartimenti, cioè (i) un vano centrale contenente cellule tumorali annegate in una recitazione gel di collagene come tumore macrospherical pseudo-primaria e (ii) un vano privo di cellule periferiche in un gel di fibrina, cioè un componente della matrice extracellulare diverso da quello utilizzato nel centro, in cui le cellule tumorali può migrare (anteriore invasione) e / o formare tumori microspherical rappresentano tumori secondari o satellitari. La formazione di tumori satellite nel compartimento periferico ènotevolmente correlata all&#39;aggressività noto o l&#39;origine metastatica delle cellule tumorali native, che rende questo sistema di coltura 3D unica. Questo approccio coltura cellulare può essere considerata per valutare invasività cancro delle cellule e la motilità, interazioni cellula-matrice e come un metodo per valutare le proprietà anti-cancro droga.

Come precedentemente accennato, una caratteristica interessante di questo test coltura cellulare 3D è che le cellule tumorali possono non solo migrare dalla spina collagene al gel di fibrina adiacente, ma anche stabilire tumori secondari (strutture ad esempio, satellite tumore-like). Questo può essere direttamente osservata con un microscopio a contrasto di fase invertito a bassi e alti ingrandimenti attraverso lo spessore di gel, in particolare con una distanza di lavoro cond...

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Three-Dimensional Cultura Assay per esplorare Cancer Cell invasività e formazione di tumori Satellite

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

three-dimensional-culture-assay-to-explore-cancer-cell-invasiveness

Research

JoVE Journal

Medicine

10.3K Views.  CHU de Québec Research Centre. Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Video: Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Spheroid Assay to Measure TGF-&#946;-induced 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 at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

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 ...

Ricerca

Medicina

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

Sistema di coltura cellulare 3D per studiare l'invasione e la valutazione terapeutica nel cancro della vescica

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Ricerca sul cancro

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Monitoraggio dell'invasione delle cellule tumorali e della citotossicità delle cellule T nella cultura 3D

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificazione del cancro al seno Cellula Invasività Utilizzando una tridimensionale (3D) Modello

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tridimensionale Co-cultura Modello di tumore stromale-Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

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 ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Cellule stromali eteromulticellulari in colture 3D prive di scaffold di cellule tumorali epiteliali per guidare l'invasione

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

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 ...

3D Culture Basement Membrane Assay: Culturing Cancer Cells on 3D Basement Membrane Protein Matrix to Study Cell Invasion

Source: Cvetkovi&#263;, D., et al. <a href="https://www.jove.com/v/51341/quantification-breast-cancer-cell-invasiveness-using-three">Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model.</a> J. Vis. Exp. (2014).
This video provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment.

Saggio della membrana basale di coltura 3D: coltura di cellule tumorali su matrice proteica della membrana basale 3D per studiare l'invasione cellulare

Source: Cvetkovi&#263;, D., et al. Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model. J. Vis. Exp. (2014).
This video ...

JoVE Encyclopedia of Experiments

Breast Cancer

In vitro studies

3-Dimensional Culture of Lung Carcinoma Cells: A Method To Study Cell-Matrix Interactions

Source: Chen, S. et. al. <a href="https://www.jove.com/v/60644/multidimensional-coculture-system-to-model-lung-squamous-carcinoma">Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression</a>. J. Vis. Exp. (2020).
This video describes the technique of 3-D culture of lung carcinoma cells to study the cell-matrix interactions. In the protocol, we will perform the 3D culture of TUM622 lung carcinoma cells to explore its potential of forming organoids in vitro.

Coltura tridimensionale di cellule di carcinoma polmonare: un metodo per studiare le interazioni cellula-matrice

Source: Chen, S. et. al. Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression. J. Vis. Exp. (2020).
This video describes the ...