Name: preparation and analysis of in vitro three dimensional breast carcinoma surrogates
Uploaded: 2016-05-09T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

The University of Alabama at Birmingham Center for Metabolic Bone Disease performed the histologic processing and sectioning of surrogates. Southern Research (Birmingham, AL) provided support for the manufacture of the bioreactor system. Funding was provided by the United States Department of Defense Breast Cancer Research Program (BC121367).

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The authors declare that they have no competing financial interests.

Qui, un metodo di coltura 3D è stato descritto che incorpora i componenti del microambiente tissutale, tra la matrice extracellulare (ECM) e fibroblasti stromali umane, in un volume che modelli più strettamente carcinoma mammario umano per consentire lo sviluppo di una morfologia 3D riepilogativo . Il metodo di coltura 3D descritto è più rappresentativo delle malattie umane di coltura cellulare in 2D tradizionale in quanto più tipi di cellule sono incorporati in un volume 3D di ECM. E &#39;stato osservato che quest...

Tridimensionale modelli (3D) cultura che imitano in modo più accurato l&#39;architettura tumorale e microambiente in vivo sono importanti per gli studi volti a sezionare le complesse interazioni tra le cellule e il loro microambiente e per testare l&#39;efficacia delle terapie candidati. Tumore impatti dimensionalità ossigeno e gradienti di nutrienti, l&#39;uniformità di esposizione al farmaco, interstiziale flusso di pressione / del sangue, e l&#39;architettura 3D 1-4. La presenza di un adeguato microambiente stromale contribuisce tumore dimensionalità e influenze segnalazione cellulare-ECM e segnalazione paracrina tra cellule stromali e cellule epiteliali maligne. Gli effetti della dimensionalità tumorale e il microambiente sulla funzione cellulare sono ben stabiliti, con entrambi i fattori che alterano la risposta ai farmaci 1,3,5-8. Inoltre, la cinetica cellulari crescita, tassi metabolici e segnalazione cellulare differiscono tra due dimensioni (2D) la cultura e la cultura in 3D, con questi fattori affecting risposta cellulare 1,3,8-10. In vitro, il microambiente tumorale surrogata può essere modulata includendo componenti ECM rappresentative e popolazioni di cellule stromali. Cellule epiteliali maligne sono influenzati dalla ECM e cellule stromali cancro-associata sia in modo sinergico / protettivo per promuovere la progressione del tumore o in maniera soppressiva per inibire ulteriormente 5,6,10 tumorale propagazione. In entrambi i contesti, lo stroma può influenzare la risposta terapeutica e somministrazione di farmaci mediante segnali paracrini e / o aumentando la pressione interstiziale nel tumore conseguente diminuzione drug delivery 1,6. Pertanto, l&#39;aggiunta di ECM e cellule stromali in modelli preclinici aiuterà Ricapitolando aspetti del tumore che non può essere modellato bene in coltura 2D. Qui un metodo per stabilire surrogati del cancro al seno che incorporano un microambiente riepilogativo, tra cui componenti ECM e scellule tromal, in un volume 3D è descritto. Nel carcinoma mammario, la popolazione di cellule stromali è prevalentemente composto di cancro associato fibroblasti (CAF) e l&#39;ECM stromale è in gran parte composto da collagene di tipo I, con una minore percentuale di componenti della matrice che si trovano nella membrana basale, tra cui laminina e collagene di tipo IV 1,4,11-13. Pertanto, questi componenti del microambiente carcinoma della mammella (cioè, CAF, collagene I, e membrana basale) sono stati incorporati nei surrogati. Questo metodo può essere utilizzato per generare solido, surrogati 3D non-perfusi (Figura 1A) o può essere adattato per includere perfusione del mezzo attraverso il surrogato tramite un sistema di bioreattore (Figura 1B). Entrambi gli approcci sono descritte qui. Questo metodo potrebbe anche essere modificato per includere altri elementi stromali, come macrofagi associati al tumore, o per modellare altri tumori solidi regolando i componenti cellulari e ECM, come appropriato. <pclass = &quot;jove_content&quot;&gt; Per il surrogato carcinoma mammario qui descritto, abbiamo utilizzato la MDA-MB-231 (231) la linea del seno delle cellule del cancro, CAF precedentemente isolato da carcinoma mammario umano 14, e un ECM composto da 90% di collagene I ( fattore di crescita materiale 6 mg / ml) e 10% ridotta membrana basale (BM). Il surrogata o è coltivato in 8 pozzetti slitta camera (surrogato solido) oppure un sistema bioreattore viene utilizzato per fornire perfusione continue dei nutrienti (surrogato perfuso). Qualsiasi sistema di perfusione bioreattore che può ospitare un volume di cellule ECM contenente può essere utilizzato 15. A titolo di esempio, si descrive la preparazione dei surrogati dei tessuti nel nostro sistema bioreattore. Questo sistema è stato sviluppato internamente e non è disponibile in commercio. Perché la nostra attenzione è rivolta alla preparazione e l&#39;analisi dei surrogati del tessuto 3D, non siamo andati in ampio dettaglio per quanto riguarda le specifiche di fabbricazione e montaggio del nostro sistema bioreattore. Tuttavia, una descrizione dettagliata diquesto sistema e il suo sviluppo è stato pubblicato 16. In questo sistema bioreattore, un canale di flusso polidimetilsilossano (PDMS) viene utilizzato per ospitare il surrogato, che è supportato da una schiuma PDMS (formato usando metodi simili a quelli descritti da Calcagnile et al. 17). Questo volume è penetrato da 4 microcanali (ogni 400 micron di diametro) che vengono continuamente perfusi da media tramite una pompa microphysiologic per la fornitura di ossigeno e sostanze nutritive per la surrogata. analisi appropriata dei surrogati è fondamentale per ottenere informazioni pertinenti per quanto riguarda la funzione cellulare in risposta al trattamento o di altre manipolazioni. Surrogati possono essere analizzati con vari metodi tra cui l&#39;imaging diretto di surrogati intatte usando la microscopia confocale o altri mezzi di imaging non invasiva, analisi cellulare indiretta analizzando i media condizionata, o perfusato, per i prodotti secreti, o analisi su sezioni istologiche dopo la fissazione e di trasformazione aparaffina. Una tale parametro che può essere valutata su sezioni istologiche è densità cellulare. Presentiamo un metodo per misurare la densità delle cellule (cioè, il numero di cellule nucleate per area sezione) utilizzando tecniche di elaborazione di immagini semi-automatizzato applicati alle microfotografie di sezioni istologiche surrogati colorate con ematossilina ed eosina (H &amp; E). La densità cellulare può essere utilizzato come indicatore della variazione relativa del numero di cellule nel tempo o risultante da diverse condizioni di crescita e trattamenti. <img alt="Figura 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> Figura volume 1. 3D e sistema di bioreattore. A) Schema del processo di generare surrogati 3D solidi. Top: cartone animato di volume di solido 3D contenente ECM (rosa), cellule di carcinoma epiteliali (di colore giallo), e CAF (arancione); In basso:. Vista dall&#39;alto di 8 pozzetti scivolo camera contenente surrogati B) Schema del processo per generare perfuso surrogati 3D. Top: cartoon di volume 3D con canali per consentire mezzo di perfusione e contenente ECM (rosa), cellule di carcinoma epiteliali (giallo), e CAF (arancione); Al centro: immagine di canale di flusso PDMS contenente PDMS schiuma (freccia nera) da iniettare con cella + ECM e penetrato da fili di acciaio inossidabile con rivestimento polimerico (freccia rosa), di 400 micron di diametro; In basso:. Immagine del canale di flusso PDMS contenente un surrogato e collegato al sistema di bioreattore per consentire continuo perfusione media (pompa peristaltica e serbatoio media non mostrato) C) Immagini di fasi di lavorazione per entrambi surrogati solidi e perfusi dopo coltura. A sinistra: immagine del cryomold contenente campioni di elaborazione gel e surrogati; Al centro: immagine di un blocco di paraffina contenente un surrogato fisso ed elaborati; A destra:. Immagine di un vetrino con una sezione istologica H &amp; E-macchiato di un surrogato <a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">Cliccate qui per visualizzare un</a>grande versione di questa figura.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagel Medium 1x (DMEM)</td><td>Corning CellGro</td><td>10-014-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>0.25% Trypsin + 2.21&amp;nbsp;mM EDTA 1x</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Tissue Culture plates, 100&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229620</td><td>Sterile</td></tr><tr><td>Tissue Culture plates, 35&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229638</td><td>For PDMS foam formation&amp;nbsp;</td></tr><tr><td>9&quot; Glass pipette</td><td>Fisher&amp;nbsp;</td><td>13-678-20D</td><td>Sterile</td></tr><tr><td>10 ml pipette</td><td>CellTreat Scientific Products</td><td>229210B</td><td>Sterile</td></tr><tr><td>1,000 &amp;micro;l piptette tips</td><td>FisherBrand</td><td>02-717-166</td><td>Sterile Filtered</td></tr><tr><td>200 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-141</td><td>Sterile Filtered</td></tr><tr><td>10 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-158</td><td>Sterile Filtered</td></tr><tr><td>15 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229410</td><td>Sterile</td></tr><tr><td>50 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229422</td><td>Sterile</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>FisherBrand</td><td>05-408-129</td><td>Sterile</td></tr><tr><td>Trypan blue</td><td>Corning Cellgro</td><td>25-900-CI</td><td>Sterile</td></tr><tr><td>Sylgard 184</td><td>Electron Microscopy Sciences</td><td>24236-10</td><td>PDMS elastomer and curing agent. Used for our in-house bioreactor.</td></tr><tr><td>PDMS Foam</td><td></td><td></td><td>Made in-house for use in our in-house bioreactor.</td></tr><tr><td>High Concentration Bovine Collagen Type I</td><td>Advanced Biomatrix</td><td>5133-A</td><td>FibriCol ~10 mg/ml</td></tr><tr><td>Growth Factor Reduced Matrigel (Basement Membrane)</td><td>Corning</td><td>354230</td><td>Basement membrane material</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S8761</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>Fisher</td><td>BP2819-1</td><td></td></tr><tr><td>DMEM 10x&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D2429</td><td></td></tr><tr><td>Nunc Lab-Tek Chamber Slide System&amp;nbsp;</td><td>Thermo Scientific</td><td>177402</td><td>8-well</td></tr><tr><td>Bioreactor</td><td></td><td></td><td>Made in-house.</td></tr><tr><td>Spring-Back 304 Stainless Steel&amp;mdash;Coated with PTFE polymer</td><td>McMaster-Carr</td><td>1749T19</td><td>Stainless steel wires to generate microchannels in our in-house&amp;nbsp; bioreactor system. 0.016&quot; Diameter</td></tr><tr><td>BioPharm Plus platinum-cured silicone pump tubing, L/S 14</td><td>Masterflex</td><td>EW-96440-14</td><td>For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in.</td></tr><tr><td>2-Stop Tubing Sets, non-flared PVC, 1.52&amp;nbsp;mm ID</td><td>Cole-Parmer&amp;nbsp;</td><td>EW-74906-36</td><td>For use in our in-house bioreactor system (with microperistalitic pump).&amp;nbsp;</td></tr><tr><td>Six Channel precision micro peristaltic pump</td><td>Cole-Parmer</td><td>EW-74906-04</td><td>For use with our in-house bioreactor system</td></tr><tr><td>Tuberculin Syringes</td><td>BD Medical</td><td>309625</td><td>26 gauge 3/8 in. needle; Sterile</td></tr><tr><td>Dissecting Tissue Forceps</td><td>FisherBrand</td><td>13-812-36</td><td>5.5 inch</td></tr><tr><td>Mini Tube Rotator</td><td>Boekel Scientific</td><td>260750</td><td>Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753)</td></tr><tr><td>50 ml tube carousel</td><td>Boekel Scientific</td><td>260753</td><td>Used with mini tube rotator</td></tr><tr><td>Bambino&amp;nbsp; Hybridization Oven</td><td>Boekel Scientific</td><td>230301</td><td>Equipment option for surrogate rotation</td></tr><tr><td>HistoGel Specimen Processing Gel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td>Specimen Processing Gel described in Step 5.2</td></tr><tr><td>Cryomold</td><td>Andwin Scientific</td><td>4566</td><td>15 mm x 15 mm x 5 mm</td></tr><tr><td>Tissue Marking Dye</td><td>Cancer Diagnostics, inc.&amp;nbsp;</td><td>03000P</td><td>Can be used to mark surrogates,&amp;nbsp; allowing multiple samples to be included in one tissue cassette&amp;nbsp;</td></tr><tr><td>Hinged tissue cassettes&amp;nbsp;</td><td>FisherBrand</td><td>22-272-416</td><td></td></tr><tr><td>Formalin</td><td>Fisher</td><td>23-245-685</td><td></td></tr><tr><td>GoldSeal Plain Glass Slides</td><td>Thermo Scientific</td><td>3048-002</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>X3P-1GAL</td><td></td></tr><tr><td>Ethanol, 200 proof (100%), USP</td><td>Decon Laboratories, Inc.</td><td>2805M</td><td></td></tr><tr><td>Hematoxylin</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7211</td><td></td></tr><tr><td>Clarifier</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7401</td><td></td></tr><tr><td>Bluing Solution</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7301</td><td></td></tr><tr><td>Eosin Y</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7111</td><td></td></tr><tr><td>Cytoseal XYL mounting media</td><td>Thermo Scientific Richard-Allan Scientific</td><td>83124</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-548-5G</td><td></td></tr></tbody></table>

preparation and analysis of in vitro three dimensional breast carcinoma surrogates

Tridimensionale (3D) cultura è un metodo più fisiologicamente rilevanti per modellare il comportamento delle cellule in vitro di due coltura dimensionale. I carcinomi, tra carcinomi mammari, sono tessuti 3D complessi composti da cellule epiteliali cancro e componenti stromali, tra cui fibroblasti e la matrice extracellulare (ECM). Tuttavia la maggior parte modelli in vitro di carcinoma mammario costituiti solo da cellule epiteliali cancerose, omettendo lo stroma e, di conseguenza, l&#39;architettura 3D di un tumore in vivo. Appropriata modellazione 3D del carcinoma è importante per la comprensione accurata della biologia del tumore, il comportamento e la risposta alla terapia. Tuttavia, la durata della cultura e volume di modelli 3D è limitata dalla disponibilità di ossigeno e nutrienti all&#39;interno della cultura. Qui, dimostriamo un metodo in cui le cellule di carcinoma mammario epiteliali e fibroblasti stromali sono incorporati in ECM per generare un surrogato cancro al seno 3D che include stroma e possono essere coltivate comestruttura 3D solido o utilizzando un sistema di bioreattore perfusione di fornire ossigeno e nutrienti. Dopo l&#39;installazione e un periodo di crescita iniziale, surrogati possono essere utilizzati per il test della droga preclinica. In alternativa, i componenti cellulari e matrice della surrogata possono essere modificati per risolvere una varietà di questioni biologiche. Dopo coltura, surrogati sono fissi ed elaborati per paraffina, in modo simile al trattamento di campioni di carcinoma mammario clinica, per la valutazione dei parametri di interesse. La valutazione di un tale parametro, la densità di cellule presenti, è spiegato, dove sono applicati ai microfotografie di sezioni istologiche di surrogati sistemi software di analisi di immagine ImageJ e CellProfiler di quantificare il numero di cellule nucleate per area. Questo può essere usato come un indicatore della variazione del numero di cellule nel tempo o la variazione del numero di cellule risultanti da diverse condizioni di crescita e trattamenti.

Entrambi surrogati di cancro al seno in 3D solidi e irrorati sono stati preparati come descritto sopra e alla produzione di 7 giorni. Successivamente, surrogati sono stati fissati, elaborati per paraffina, sezionati e colorati con ematossilina ed eosina, come descritto sopra. Il numero di cellule nucleate per area (sia 231 cellule e CAF) di ciascun surrogata è stata misurata. Come si vede nella figura 12, microfotografie rappresentativi delle sezioni H colorati con EE d...

Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Preparazione e analisi di<em&gt; In Vitro</em&gt; Tridimensionale carcinoma mammario mondo dei replicanti

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

preparation-analysis-vitro-three-dimensional-breast-carcinoma

Research

JoVE Journal

Bioengineering

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

10.6K Views.  University of Alabama at Birmingham. We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Video: Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

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

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

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

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

Ricerca

Ricerca sul cancro

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Istituzione e analisi di organidi tridimensionali (3D) Derivati dagli spettri della metastasi ossea del cancro della prostata del paziente e dai loro xenoinfi

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Isolamento e valutazione funzionale di cellule staminali di cancro al seno umano da campioni di cellule e tessuti

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Modello biomimetico tridimensionale in vitro di neuroblastoma utilizzando scaffold a base di collagene

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Sferoidi epiteliali ed endoteliali mammari come potenziale modello funzionale in vitro per la ricerca sul cancro al seno

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

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 ...

Medicina

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Metodi per la coltura di femore umano espianti di tessuto di studio sul carcinoma della mammella cellulare colonizzazione della nicchia metastatico

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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 ...

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

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

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 ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

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