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.

En este documento, un método de cultivo 3D se ha descrito que incorpora componentes del microambiente del tejido, incluyendo la matriz extracelular (ECM) y fibroblastos estromales humanos, en un volumen que más de cerca los modelos de cáncer de mama humano para permitir el desarrollo de una morfología 3D recapitulativo . El método de cultivo 3D descrito es más representativo de la enfermedad humana de cultivo de células 2D tradicional en que múltiples tipos de células se incorporan en un volumen 3D de ECM. Se h...

Tres modelos tridimensionales (3D) de cultivo que imitan con mayor precisión la arquitectura tumor y microentorno in vivo son importantes para los estudios dirigidos a diseccionar las complejas interacciones entre las células y su microambiente y para probar la eficacia de las terapias candidatos. Tumor impactos dimensionalidad de oxígeno y los gradientes de nutrientes, la uniformidad de la exposición al fármaco, el flujo / presión arterial intersticial, y la arquitectura 3D 1-4. La presencia de un microambiente estromal apropiado contribuye a la dimensionalidad tumor y las influencias señalización celular-ECM y la señalización paracrina entre las células del estroma y las células epiteliales malignas. Los efectos de la dimensionalidad del tumor y el microambiente de la función celular están bien establecidos, con los dos factores que alteran la respuesta a los fármacos 1,3,5-8. Además, la cinética de crecimiento celular, las tasas metabólicas y de señalización celular difieren entre las dos culturas dimensiones (2D) y la cultura en 3D, con estos factores affenexión respuesta celular 1,3,8-10. In vitro, el microambiente sustituto tumor puede ser modulada mediante la inclusión de componentes de ECM representativos y las poblaciones de células del estroma. Células epiteliales malignos son influenciados por el ECM y las células del estroma asociadas con el cáncer, ya sea de una manera sinérgica / protección para promover la progresión tumoral o de una manera supresora para inhibir aún más la propagación 5,6,10 tumor. En cualquier contexto, el estroma puede afectar a la respuesta terapéutica y la administración de fármacos a través de la señalización paracrina y / o mediante el aumento de la presión intersticial en el tumor resulta en una disminución de suministro de fármaco de 1,6. Por lo tanto, la adición de ECM y las células del estroma en modelos preclínicos ayudará recapitular aspectos del tumor que no puede ser modelado bien en cultivo 2D. En este documento un método para establecer sustitutos de cáncer de mama que incorporan un microambiente recapitulativo, incluyendo constituyentes y s ECMtromal células, en un volumen 3D se describe. En el carcinoma de mama, la población de células del estroma se predominantemente compuesta de cáncer asociado fibroblastos (CAF) y el ECM del estroma se compone principalmente de colágeno de tipo I con una menor proporción de componentes de la matriz que se encuentran en la membrana basal, incluyendo laminina y colágeno tipo IV 1,4,11-13. Por lo tanto, estos componentes del microambiente carcinoma de mama (es decir, CAF, colágeno I, y la membrana basal) se han incorporado en los sustitutos. Este método puede ser utilizado para generar sólidos, sustitutos 3D-un perfundido (Figura 1A) o puede ser adaptado para incluir la perfusión de medio a través de la madre sustituta a través de un sistema de biorreactor (Figura 1B). Ambos enfoques se describen aquí. Este método también podría ser modificado para incluir otros elementos del estroma, tales como macrófagos asociados al tumor, o para modelar otros tumores sólidos mediante el ajuste de los componentes celulares y de ECM, como sea apropiado. <pclass = &quot;jove_content&quot;&gt; Para el sustituto carcinoma de mama describe aquí, hemos utilizado el MDA-MB-231 (231) línea celular de cáncer de mama, la CAF previamente aislado de carcinoma de mama humano 14, y un ECM compuesto por 90% de colágeno I ( factor de crecimiento de material de 6 mg / ml) y 10% de reducción de la membrana basal (BM). El sustituto se cultiva bien en un 8 pocillos diapositiva cámara (sustituto sólido) o un sistema de biorreactor se utiliza para proporcionar la perfusión de nutrientes continua (sustituto perfundido). Cualquier sistema de biorreactor de perfusión que puede alojar un volumen de células que contiene ECM se puede utilizar 15. Como ejemplo, se describe la preparación de los sustitutos de tejido en nuestro sistema de biorreactor. Este sistema fue desarrollado internamente y no está disponible comercialmente. Debido a que nuestra atención se centra en la preparación y el análisis de los sustitutos de tejido 3D, no hemos entrado en una descripción más detallada con respecto a los detalles de fabricación y montaje de nuestro sistema de biorreactor. Sin embargo, una descripción detallada deeste sistema y su desarrollo ha sido publicado el 16. En este sistema de biorreactor, un canal de flujo de polidimetilsiloxano (PDMS) se utiliza para alojar el sustituto, que está soportado por una espuma de PDMS (formado usando métodos similares a los descritos por Calcagnile et al. 17). Este volumen es penetrado por 4 microcanales (cada uno de 400 m de diámetro) que son perfundidos continuamente por medio de una bomba a través de microphysiologic para suministrar oxígeno y nutrientes a la madre sustituta. análisis apropiado de los sustitutos es crucial para obtener la información pertinente a la función celular en respuesta al tratamiento u otras manipulaciones. Los sustitutos pueden ser analizadas por diversos métodos, incluyendo formación de imágenes directa de los sustitutos intactas mediante microscopía confocal o de otros medios de formación de imágenes no invasiva, análisis celular indirecto mediante el ensayo de los medios acondicionados, o perfundido, para los productos secretados, o análisis en cortes histológicos después de la fijación y el procesamiento aparafina. Uno de estos parámetros que pueden ser evaluados en los cortes histológicos es la densidad celular. Presentamos un método para medir la densidad de células (es decir, el número de células nucleadas por área de la sección), utilizando técnicas de procesamiento de imagen semi-automatizados aplicados a fotomicrografías de secciones histológicas de sustitución teñidas con hematoxilina y eosina (H &amp; E). La densidad celular se puede utilizar como un indicador del cambio relativo en el número de células en el tiempo o que resulta de diferentes condiciones de crecimiento y tratamientos. <img alt="Figura 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> Figura 1. volumen 3D y el sistema de biorreactor. A) Representación esquemática del proceso para generar sustitutos 3D sólidos. Arriba: dibujos animados de volumen 3D sólido que contiene ECM (rosa), células de carcinoma epitelial (amarillo), y la CAF (naranja); En pocas palabras:. Vista desde arriba de sustitutos de 8 pocillos de diapositivas cámara que contiene B) Esquema del proceso para generar una perfusión sustitutos 3D. Arriba: cartoon de volumen 3D con canales para permitir la perfusión del medio y que contiene ECM (rosa), carcinoma de células epiteliales (amarillo), y la CAF (naranja); Medio: imagen del canal de flujo de PDMS que contiene PDMS espuma (flecha negro) para ser inyectado con células + ECM y penetrado por alambres de acero inoxidable recubiertos de polímero (flecha rosa) que miden 400 micras de diámetro; Inferior:. Imagen del canal de flujo PDMS que contiene un sustituto y conectado al sistema de biorreactor para permitir la perfusión del medio continuo (bomba peristáltica y depósito de medio que no se muestra) C) Imágenes de pasos de procesamiento para ambos sustitutos sólidos y perfundidos después del cultivo. A la izquierda: imagen de la criomolde que contiene la muestra de procesamiento de gel y sustituta; Medio: imagen de un bloque de parafina que contiene un sustituto fijo y procesados; Derecha:. Imagen de una placa de vidrio con un corte histológico teñido con HE y de un sustituto <a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">Haga clic aquí para ver una</a>versión más grande de esta 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

En tres dimensiones (3D) la cultura es un método más fisiológicamente relevantes para modelar el comportamiento celular in vitro de dos dimensiones la cultura. Los carcinomas, incluyendo los carcinomas de mama, son tejidos 3D complejas compuestas de células epiteliales de cáncer y los componentes del estroma, incluyendo fibroblastos y matriz extracelular (ECM). Sin embargo, la mayoría de los modelos in vitro de carcinoma de mama consisten solamente de las células epiteliales cancerosas, omitiendo el estroma y, por lo tanto, la arquitectura 3D de un tumor in vivo. modelado 3D apropiada del carcinoma es importante para la comprensión exacta de la biología del tumor, el comportamiento y la respuesta al tratamiento. Sin embargo, la duración de la cultura y el volumen de los modelos 3D está limitada por la disponibilidad de oxígeno y nutrientes en el cultivo. En este documento, se demuestra un método en el que las células de carcinoma de mama epiteliales y los fibroblastos del estroma se incorporan en ECM para generar un sustituto cáncer de mama en 3D que incluye estroma y se pueden cultivar como unaestructura 3D sólida o mediante el uso de un sistema de biorreactor de perfusión para entregar oxígeno y nutrientes. Después de la instalación y un período de crecimiento inicial, sustitutos pueden ser utilizados para las pruebas de drogas preclínica. Alternativamente, los componentes celulares y de la matriz de la madre sustituta pueden ser modificadas para abordar una variedad de cuestiones biológicas. Después del cultivo, sustitutos se fijaron y procesaron para parafina, de una manera similar a la manipulación de las muestras de carcinoma de mama clínica, para la evaluación de los parámetros de interés. La evaluación de uno de estos parámetros, la densidad de células presentes, se explica, en donde sistemas de software de análisis de imagen ImageJ y CellProfiler se aplican a fotomicrografías de secciones histológicas de sustitutos para cuantificar el número de células nucleadas por área. Esto se puede utilizar como un indicador del cambio en el número de células en el tiempo o el cambio en el número de células como resultado de diferentes condiciones de crecimiento y tratamientos.

Tanto sustitutos de cáncer de mama sólidos y perfundidos 3D se prepararon como se ha descrito anteriormente y se cultivaron durante 7 días. Posteriormente, sustitutos se fijaron, se procesaron para parafina, seccionadas y teñidas con hematoxilina y eosina, tal como se describe anteriormente. Se midió el número de células nucleadas por área (ambas células 231 y CAF) de cada sustituto. Como puede verse en la Figura 12, fotomicrografías representativas de las secc...

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.

Preparación y análisis de<em&gt; In Vitro</em&gt; Tres de mama Carcinoma de dimensiones 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 vistas.  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.

Estudio del triple cáncer de mama negativo con el modelo de cáncer de mama ortotópico

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

Investigación

Investigación sobre el cáncer

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.

Establecimiento y Análisis de Organoides Tridimensionales (3D) Derivados de los Especímenes de Metastasis ósea del Cáncer de Próstata del Paciente y sus Xenoinjertos

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.

Aislamiento y evaluación funcional de células madre de cáncer de mama humano a partir de muestras de células y tejidos

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.

Modelo biomimético tridimensional in vitro de neuroblastoma con andamios basados en colágeno

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.

Esferoides epiteliales y endoteliales mamarios como un modelo funcional potencial in vitro para la investigación del cáncer de mama

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.

La cuantificación de la invasividad de las células del cáncer de mama El uso de un modelo (3D) Tres Dimensiones

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.

Métodos de explantes de tejidos El cultivo fémur humano para el Estudio de la colonización de la célula del cáncer de pecho de la Niche metastásico

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.

Tumor Ensayo tridimensional (3D) esferoide Invasion

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.

Tridimensional Ensayo de Cultivo para explorar invasividad de las células del cáncer y la formación de tumores satélite

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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