Name: preparation and analysis of in vitro three dimensional breast carcinoma surrogates
Uploaded: 2016-05-09T15:00:00.000+00:00
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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.

Ici, un procédé de culture en 3D a été décrite qui incorpore les composants du micro-environnement tissulaire, y compris la matrice extracellulaire (ECM) et les fibroblastes du stroma de l&#39;homme, dans un volume plus près des modèles de cancer du sein humain pour permettre le développement d&#39;une morphologie en 3D récapitulatif . Le procédé de culture en 3D décrit est plus représentatif de la maladie chez l&#39;homme que la culture cellulaire traditionnelle 2D en ce que plusieurs types de cellules son...

Trois modèles (3D) de culture de dimensions qui imitent plus précisément l&#39;architecture de la tumeur et microenvironnement in vivo sont importantes pour des études visant à disséquer les interactions complexes entre les cellules et leur microenvironnement et pour tester l&#39;efficacité des thérapies candidates. Tumeur impacts dimensionnalité de l&#39; oxygène et des gradients de nutriments, l&#39;uniformité de l&#39; exposition au médicament, interstitielle débit / pression sanguine, et de l&#39; architecture 3D 1-4. La présence d&#39;un micro-environnement stromal approprié contribue à la dimensionnalité des tumeurs et des influences de signalisation cellule-ECM et la signalisation entre paracrine des cellules stromales et les cellules épithéliales malignes. Les effets de la dimensionnalité de la tumeur et le microenvironnement sur ​​la fonction cellulaire sont bien établis, avec les deux facteurs altérant la réponse aux médicaments 1,3,5-8. En outre, la cinétique cellulaire de croissance, les taux métaboliques, et la signalisation cellulaire diffèrent entre les deux (2D) la culture et de la culture dimensionnelle en 3D, avec ces facteurs affection réponse cellulaire 1,3,8-10. In vitro, la tumeur microenvironnement porteuse peut être modulée en incluant des composantes représentatives de la MEC et les populations de cellules stromales. Les cellules épithéliales malignes sont influencées par l&#39;ECM et les cellules stromales associées au cancer soit d&#39;une manière synergique / protecteur pour favoriser la progression tumorale ou d&#39;une manière suppressive pour inhiber la propagation ultérieure de la tumeur 5,6,10. Dans les deux contexte, le stroma peut affecter la réponse thérapeutique et la délivrance de médicaments par l&#39; intermédiaire d&#39;une signalisation paracrine et / ou en augmentant la pression interstitielle dans la tumeur , entraînant une diminution de l&#39; administration de médicaments à 1,6. Par conséquent, l&#39;ajout de l&#39;ECM et les cellules stromales dans des modèles précliniques aidera récapituler les aspects de la tumeur qui ne peut pas être bien modélisé dans la culture 2D. Ici une méthode pour établir des substituts du cancer du sein qui incorporent un microenvironnement récapitulatifs, y compris les composants et s ECMtromal cellules dans un volume 3D est décrit. Dans le cancer du sein, la population de cellules stromales est principalement constitué de fibroblastes associés au cancer (FAC) et l&#39;ECM stromales se compose essentiellement de collagène de type I avec une faible proportion de composants de la matrice que l&#39;on trouve dans la membrane basale, y compris la laminine et du collagène de type IV 1,4,11-13. Par conséquent, ces composants du microenvironnement de carcinome du sein (c. -à- CAF, le collagène I, et la membrane basale) ont été incorporés dans les mères porteuses. Cette méthode peut être utilisée pour générer des solides, des substituts de l&#39; ONU 3D perfusé (figure 1A) ou peut être adapté pour inclure la perfusion du milieu à travers le substitut par l&#39; intermédiaire d&#39; un système de bioréacteur (figure 1B). Les deux approches sont décrites ici. Cette méthode peut également être modifiée pour inclure d&#39;autres éléments du stroma, telles que les macrophages associés aux tumeurs, ou pour modéliser d&#39;autres tumeurs solides en ajustant les composants cellulaires et ECM, le cas échéant. <pclass = &quot;jove_content&quot;&gt; Pour la substitution de carcinome du sein décrit ici, nous avons utilisé la MDA-MB-231 (231) , la lignée cellulaire de cancer du sein, CAF précédemment isolé du carcinome du sein humain 14, et un ECM composé de 90% de collagène I ( le facteur de croissance matériau 6 mg / ml) et 10% de sous-sol réduite membrane (BM). Le substitut est soit cultivé dans un 8 puits lame de chambre (substitut solide) ou un système de bioréacteur est utilisé pour fournir une perfusion de nutriment continue (substitution perfusé). Tout système de bioréacteur à perfusion qui peut accueillir un volume de cellules ECM contenant peut être utilisé 15. A titre d&#39;exemple, nous décrivons la préparation des succédanés de tissus dans le système de bioréacteur. Ce système a été développé en interne et ne sont pas disponibles dans le commerce. Parce que notre accent est mis sur la préparation et l&#39;analyse des substituts de tissu 3D, nous ne sommes pas allés dans de nombreux détails sur les spécificités de la fabrication et l&#39;assemblage de notre système de bioréacteur. Toutefois, une description détailléece système et son développement a été publié le 16. Dans ce système de bioréacteur, un canal d&#39;écoulement (PDMS) est utilisé pour loger la porteuse, qui est supporté par une mousse de PDMS (formé en utilisant des procédés similaires à ceux décrits par Calcagnile et al. 17). Ce volume est traversé par des microcanaux 4 (chacun de 400 um de diamètre) qui sont continuellement perfusées par du milieu par l&#39;intermédiaire d&#39;une pompe à microphysiologic pour fournir de l&#39;oxygène et des nutriments à la substitution. Une analyse appropriée des mères porteuses est crucial d&#39;obtenir des informations pertinentes concernant la fonction cellulaire en réponse à un traitement ou d&#39;autres manipulations. Les substituts peuvent être analysés par différents procédés, y compris l&#39;imagerie directe des mères porteuses intactes par microscopie confocale ou d&#39;autres moyens d&#39;imagerie non invasive, l&#39;analyse cellulaire indirecte par dosage du milieu conditionné ou perfusé, pour les produits sécrétés, ou des analyses sur des coupes histologiques après la fixation et le traitement àparaffine. Un tel paramètre qui peut être évalué sur des coupes histologiques est la densité cellulaire. Nous présentons une approche pour mesurer la densité cellulaire ( à savoir, le nombre de cellules nucléées par section zone) en utilisant des techniques de traitement d&#39; image semi-automatisés appliqués aux photomicrographies des coupes histologiques de substitution colorées à l&#39; hématoxyline et l&#39; éosine (H &amp; E). La densité cellulaire peut être utilisée comme un indicateur de la variation relative du nombre de cellules au cours du temps ou qui résulte de différentes conditions de croissance et les traitements. <img alt="Figure 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> Figure 1. Volume 3D et système de bioréacteur. A) Schéma du processus pour produire des substituts 3D solides. Top: bande dessinée de volume 3D solide contenant ECM (rose), des cellules de carcinome épithéliales (jaune) et CAF (orange); Bottom:. Vue de dessus de 8 puits chambre diapositive contenant des substituts B) Schéma du processus pour générer perfusé mères porteuses 3D. Top: cartoon de volume 3D avec des canaux pour permettre perfusion de milieu et contenant ECM (rose), des cellules de carcinome épithéliales (jaune) et CAF (orange); Moyen: image de canal d&#39;écoulement PDMS contenant de la mousse PDMS (flèche noire) à injecter avec la cellule + ECM et pénétrée par des fils d&#39;acier inoxydable enduites de polymère (flèche rose) mesurant 400 m de diamètre; En bas:. L&#39; image du canal d&#39;écoulement de PDMS contenant une mère porteuse et connecté au système de bioréacteur pour permettre une perfusion de milieu continu (pompe péristaltique et le réservoir de support non représenté) , C) , les images des étapes de traitement pour les mères porteuses solides et perfusées après la culture. Gauche: image du cryomoule contenant l&#39;échantillon de traitement du gel et de substitution; Moyen: l&#39;image d&#39;un bloc de paraffine contenant un substitut fixe et traité; A droite:. Image d&#39;une lame de verre avec une coupe histologique H &amp; E teinté d&#39;un substitut <a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une</a>une plus grande version de ce chiffre.

<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

Trois dimensions de la culture (3D) est une méthode plus physiologiquement pertinents pour modéliser le comportement des cellules in vitro que deux cultures dimensionnel. Les cancers, y compris les cancers du sein sont des tissus 3D complexes composées de cellules cancéreuses épithéliales et stromales de composants, y compris les fibroblastes et la matrice extracellulaire (ECM). Mais la plupart des modèles in vitro de cancer du sein sont constitués uniquement de cellules epitheliales cancéreuses, en omettant le stroma et, par conséquent, l&#39;architecture en 3D d&#39;une tumeur in vivo. modélisation 3D appropriée du cancer est important pour la compréhension précise de la biologie de la tumeur, le comportement et la réponse au traitement. Toutefois, la durée de la culture et le volume des modèles 3D est limitée par la disponibilité de l&#39;oxygène et des nutriments dans la culture. Ici, nous démontrons une méthode dans laquelle les cellules de carcinome du sein épithéliales et les fibroblastes du stroma sont incorporés dans l&#39;ECM pour générer une porteuse de cancer du sein en 3D qui comprend stroma et peuvent être cultivées en tant questructure 3D solide ou en utilisant un système de bioréacteur à perfusion pour délivrer de l&#39;oxygène et de nutriments. Après l&#39;installation et une période de croissance initiale, les substituts peuvent être utilisés pour le dépistage des drogues préclinique. En variante, les composants cellulaires et de la matrice de la mère porteuse peuvent être modifiés pour répondre à une variété de questions biologiques. Après la culture, les mères porteuses sont fixées et traitées à la paraffine, d&#39;une manière similaire à la manipulation d&#39;échantillons de carcinome du sein clinique pour l&#39;évaluation des paramètres d&#39;intérêt. L&#39;évaluation de l&#39;un de ces paramètres, la densité de cellules présentes, il est expliqué, le cas des systèmes logiciels d&#39;analyse d&#39;image et ImageJ CellProfiler sont appliqués à des photomicrographies des coupes histologiques de mères porteuses pour quantifier le nombre de cellules nucléées par zone. Ceci peut être utilisé comme un indicateur de la variation du nombre de cellules au cours du temps ou la variation du nombre de cellules résultant de différentes conditions de croissance et les traitements.

Deux 3D mères porteuses de cancer du sein solides et perfusées ont été préparées comme décrit ci-dessus et cultivées pendant 7 jours. Par la suite, les mères porteuses sont fixées, traitées à la paraffine, sectionnées et colorées avec de l&#39;hématoxyline et de l&#39;éosine comme décrit ci-dessus. Le nombre de cellules nucléées par zone (les deux cellules 231 et CAF) de chaque porteuse a été mesurée. Comme on peut le voir sur la figure 12, photomic...

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.

Préparation et analyse des<em&gt; In vitro</em&gt; Trois Breast Dimensional Carcinome Surrogates

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 vues.  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.

Vidéo: 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.

Étudier le cancer du sein triple négatif à l’aide d’un modèle orthotopique de cancer du sein

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

Recherche

Recherche en cancérologie

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.

Établissement et analyse des organoïdes tridimensionnels (3D) dérivés des spécimens de métastasie osseuse du cancer de la prostate du patient et de leurs Xénogreffes

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.

Isolement et évaluation fonctionnelle des cellules souches du cancer du sein humain à partir d’échantillons de cellules et de tissus

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.

Modèle biomimétique in vitro tridimensionnel de neuroblastome à l’aide d’échafaudages à base de collagène

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.

Les sphéroïdes épithéliaux et endothéliaux mammaires comme modèle fonctionnel in vitro potentiel pour la recherche sur le cancer du sein

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.

Quantification du cancer du sein cellulaire envahissement aide d&#39;un (3D) du modèle en trois dimensions

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

Médecine

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.

Méthodes pour explants de tissus en culture fémur humain pour étudier le cancer du sein cellulaire colonisation de la Niche métastatique

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.

Tridimensionnelle (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 ...

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 Culture Assay pour explorer des cellules cancéreuses invasives et Tumor Satellite Formation

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.

Préparation et analyse des

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