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).

<ol>
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The authors declare that they have no competing financial interests.

Hierin wurde ein Verfahren der 3D-Kultur beschrieben, die Komponenten des Gewebemikroumgebung beinhaltet, einschließlich der extrazellulären Matrix (ECM) und menschliche Stromazellen Fibroblasten, in einem Volumen, das genauer Modelle menschlichen Brustkrebs für die Entwicklung eines zusammenfassenden 3D-Morphologie zu ermöglichen, . Die 3D-Kultivierungsverfahren beschrieben ist repräsentativer für menschliche Krankheiten als herkömmliche 2D-Zellkultur, dass mehrere Zelltypen werden in einem 3D-Volumen von ECM in...

Dreidimensionale (3D) Kulturmodelle , die genauer den Tumor Architektur und Mikroumgebung in vivo nachahmen sind wichtig für die Studien mit dem Ziel , die komplexen Wechselwirkungen zwischen Zellen und ihrer Mikroumgebung zu sezieren und die Wirksamkeit von Therapien Kandidaten zu testen. Tumor Dimensionalität Auswirkungen von Nährstoffen und Sauerstoff - Gradienten, die Gleichförmigkeit der Wirkstoffexposition, interstitielle Druck / Durchblutung und 3D - Architektur 1-4. Die Anwesenheit eines geeigneten stromalen Mikroumgebung trägt zur Tumor Dimensionalität und Einflüsse zell ECM Signalisierungs- und parakrine Signalisierung zwischen Stromazellen und maligne Epithelzellen. Die Auswirkungen von Tumor Dimensionalität und der Mikroumgebung auf die Zellfunktion sind gut etabliert, mit beiden Faktoren verändern Arzneimittelreaktion 1,3,5-8. Zusätzlich Zellwachstum Kinetik, Stoffwechselraten und Zellsignalisierung unterscheiden sich zwischen den zweidimensionalen (2D) Kultur und Kultur in 3D, mit diesen Faktoren affefesselndes zelluläre Antwort 1,3,8-10. In vitro kann der Tumor - Mikroumgebung Surrogat indem repräsentative ECM Bestandteile und Stroma - Zellpopulationen moduliert werden. Maligne Epithelzellen werden durch das ECM und Krebs-assoziiertes Stroma - Zellen , die entweder in einer synergistischen / protektiv Weise beeinflusst Tumorprogression oder in einer unterdrückenden Art und Weise weiter zu fördern Tumorausbreitung 5,6,10 hemmen. In jedem Zusammenhang kann das Stroma beeinflussen therapeutische Reaktion und Arzneimittelabgabe über parakrine Signalisierungs- und / oder durch interstitiellen Druck in dem Tumor Erhöhung was zu einer verringerten Medikamentenabgabe 1,6. Daher hilft die Zugabe von ECM und Stromazellen in präklinischen Modellen Aspekte des Tumors rekapitulieren, die nicht gut in 2D Kultur modelliert werden können. Hierin wird ein Verfahren Brustkrebs Surrogate zu etablieren, die eine zusammenfassender Mikro integrieren, einschließlich ECM Bestandteile und stromal Zellen in einem 3D-Volumen beschrieben. In Brustkarzinom ist die stromale Zellpopulation überwiegend wird Krebs-Fibroblasten (CAF) und dem Stroma ECM besteht aus weitgehend von Kollagen Typ zusammengesetzt I mit einem kleineren Anteil der Matrixkomponenten, die in der Basalmembran gefunden werden, einschließlich Laminin und Kollagen Typ IV 1,4,11-13. Daher werden diese Komponenten des Mammakarzinoms Mikroumgebung (dh, CAF, Kollagen I und Basalmembran) wurden in die Surrogate eingebaut. Dieses Verfahren kann verwendet werden , um feste, nicht-perfundierten 3D Surrogate (1A) zu erzeugen , oder können angepasst werden Perfusion des Mediums durch das Surrogat über ein Bioreaktorsystem (1B) aufzunehmen. Beide Ansätze werden hier beschrieben. Dieses Verfahren könnte auch auf andere stromalen Elemente modifiziert werden, wie beispielsweise Tumor-assoziierten Makrophagen, oder andere feste Tumore zu modellieren, indem die zellulären und ECM-Komponenten, gegebenenfalls anzupassen. <pclass = &quot;jove_content&quot;&gt; Für das Surrogat Mammakarzinoms hier beschrieben, haben wir die MDA-MB-231 (231) Brustkrebs-Zelllinie, CAF zuvor von menschlichen Brustkarzinom 14 und eine ECM , bestehend aus 90% Kollagen I isoliert verwendet ( 6 mg / ml) und 10% Wachstumsfaktor reduziert Basalmembran Material (BM). Der Ersatz wird in einem 8-Well-Kammerobjektträger (fest Surrogat) oder ein Bioreaktorsystem verwendet wird, um entweder angebaut kontinuierliche Nährstoff Perfusion zu liefern (durchbluteten Surrogat). Jede Perfusions - Bioreaktor - System , das ein Volumen von ECM enthaltenden Zellen aufnehmen kann , kann 15 verwendet werden. Als Beispiel beschreiben wir die Herstellung der Gewebe Surrogate im Bioreaktorsystem. Dieses System wurde im eigenen Haus entwickelt und ist nicht im Handel erhältlich. Weil unsere hier konzentrieren sich auf die Vorbereitung und Analyse der 3D-Gewebe Surrogate ist, haben wir nicht in umfangreichen Detail in Bezug auf die Besonderheiten der Herstellung und Montage unserer Bioreaktorsystem gegangen. Jedoch wird eine detaillierte Beschreibung vonDieses System und seine Entwicklung wurde 16 veröffentlicht. In diesem Bioreaktorsystem, ein Polydimethylsiloxan (PDMS) Strömungskanal des Surrogat zu Haus verwendet wird, die von einem PDMS - Schaum unterstützt wird (gebildet Methoden ähnlich denen von Calcagnile beschriebenen et al. 17). Dieses Volumen wird von 4 Mikrokanäle (jeweils 400 &amp; mgr; m im Durchmesser) durchsetzt, die durch Medium über eine microphysiologic Pumpe kontinuierlich durchblutet werden Sauerstoff und Nährstoffe zum Ersatz zu liefern. Entsprechende Analyse der Surrogate ist entscheidend wichtige Informationen in Bezug auf die Zellfunktion in Reaktion auf die Behandlung oder andere Manipulationen zu gewinnen. Surrogates kann durch verschiedene Verfahren einschließlich der direkten Bildgebung von intaktem Surrogate mittels konfokaler Mikroskopie oder andere Mittel der nichtinvasiven Bildgebung, indirekte Zellanalyse durch Bestimmung des konditionierten Medien oder Perfusat, für sezernierte Produkte oder Analyse auf histologischen Schnitten nach der Fixierung und Verarbeitung analysiert werden, nachParaffin. Ein solcher Parameter, der auf histologischen Schnitten ausgewertet werden kann, ist die Zelldichte. Wir stellen einen Ansatz Zelldichte zu messen (dh die Anzahl von nukleierten Zellen pro Querschnittsfläche) unter Verwendung von halbautomatischen Bildverarbeitungstechniken auf Mikrophotographien von Surrogat histologischen Schnitte angewendet gefärbt mit Hämatoxylin und Eosin (H &amp; E). Die Zelldichte kann als ein Indikator der relativen Änderung der Zellzahl über die Zeit verwendet werden, oder dass die Ergebnisse von unterschiedlichen Wachstumsbedingungen und Behandlungen. <img alt="Abbildung 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> Abbildung 1. 3D - Volumen und Bioreaktorsystem. A) Schematische Darstellung des Prozesses solide 3D ​​- Surrogate zu erzeugen. Top: Karikatur von festen 3D-Volumen enthalten, ECM (pink), epithelialen Karzinomzellen (gelb) und CAF (orange); Unten:. Draufsicht auf 8-Well - Kammer - Folie mit Surrogaten B) Schematische Darstellung des Prozesses 3D Surrogate durchbluteten zu erzeugen. Top: cartoon von 3D-Volumen mit Kanälen für mittlere Perfusion und enthält ECM (pink), epithelialen Karzinomzellen (gelb) und CAF (orange) zu ermöglichen; Mitte: Bild von PDMS Strömungskanal enthält PDMS Schaum (schwarzer Pfeil), um mit Zelle injiziert + ECM und drang durch polymerbeschichteten rostfreien Stahldrähten (pink Pfeil) Messung 400 &amp; mgr; m im Durchmesser; Unten:. Bild des PDMS Strömungskanal einen Surrogat und mit dem Bioreaktorsystem zu ermöglichen , für die kontinuierliche Mediumperfusion (Schlauchpumpe und Medienspeicher nicht dargestellt) C) Bilder von Verarbeitungsschritten für sowohl feste als auch perfundiert Surrogate nach Kultur enthält. Links: Bild des cryomold Probenverarbeitung Gel und Surrogat enthält; Mitte: Bild eines Paraffinblock eine feste und verarbeitet Surrogat enthält; Rechts:. Bild von einem Glasträger mit einem H &amp; E-gefärbten histologischen Schnitt eines Ersatz <a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">Bitte hier klicken</a> , <a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">um anzuzeigen</a>Größere Version der Figur.

<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

Dreidimensionale (3D) Kultur ist eine physiologisch relevante Methode Zellverhalten in vitro als zweidimensionale Kultur zu modellieren. Karzinome, Brustkarzinome, einschließlich, sind komplexe 3D-Gewebe von Krebs besteht Epithelzellen und Stromazellen Komponenten, einschließlich Fibroblasten und extrazelluläre Matrix (ECM). Doch die meisten in vitro - Modelle von Mammakarzinom nur von Krebs epithelialen Zellen bestehen, Weglassen des Stromas und damit die 3D - Architektur eines Tumors in vivo. Entsprechende 3D-Modellierung von Karzinomen ist wichtig für eine genaue Verständnis der Tumorbiologie, Verhalten und Ansprechen auf die Therapie. Jedoch wird die Dauer der Kultur und das Volumen von 3D-Modellen durch die Verfügbarkeit von Sauerstoff und Nährstoffe in der Kultur beschränkt. Hierin zeigen wir ein Verfahren, bei dem Mammakarzinom Epithelzellen und Stromazellen Fibroblasten in ECM integriert sind ein 3D Brustkrebs Surrogat zu erzeugen, das Stroma enthält und als kultiviert werden,solide 3D-Struktur oder durch eine Perfusionsbioreaktorsystem System unter Verwendung von Sauerstoff und Nährstoffe zu liefern. Nach Einrichtung und einer anfänglichen Wachstumsperiode Surrogate können für die präklinische Drogentests verwendet werden. Alternativ können die zelluläre und Matrixkomponenten des Surrogat modifiziert werden, um eine Vielzahl von biologischen Fragen anzugehen. Nach der Kultur werden fixiert und Surrogate, in einer Weise zur Auswertung von Parametern von Interesse für die Behandlung der klinischen Brustkarzinom Proben, ähnlich zu Paraffin verarbeitet. Die Auswertung eines solchen Parameters, der Dichte der Zellen vorhanden ist, erklärt, wobei ImageJ und CellProfiler Bildanalysesoftwaresysteme Photomikrographien von histologischen Schnitten von Surrogaten angewendet werden, um die Anzahl von nukleierten Zellen pro Fläche zu quantifizieren. Dies kann als ein Indikator für die Veränderung der Zellzahl über die Zeit oder der Veränderung der Zellzahl verwendet werden, die aus unterschiedlichen Wachstumsbedingungen und Behandlungen.

Sowohl feste und durchbluteten Surrogate 3D Brustkrebs wurden wie oben beschrieben hergestellt und gezüchtet für 7 Tage. Anschließend wurden Surrogate fixiert, verarbeitet zu Paraffin, geschnitten und gefärbt mit Hämatoxylin und Eosin, wie oben beschrieben. Die Zahl der kernhaltigen Zellen pro Fläche (beide 231-Zellen und CAF) jedes Surrogat wurde gemessen. Wie in Figur 12 repräsentativ Photomikrographien der H &amp; E-gefärbten Schnitten zeigen eine höhere Konz...

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.

Herstellung und Analyse von<em&gt; In Vitro</em&gt; Three Dimensional Mammakarzinoms 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 Aufrufe.  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.

Serie: 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.

Studieren von dreifach negativem Brustkrebs mit orthotopischem Brustkrebsmodell

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

Forschung

Krebsforschung

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.

Etablierung und Analyse von dreidimensionalen (3D) Organoiden, abgeleitet von Patienten prostatakrebs Knochenmetastasen Proben und ihre Xenografts

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.

Isolierung und funktionelle Beurteilung menschlicher Brustkrebsstammzellen aus Zell- und Gewebeproben

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.

Dreidimensionales biomimetisches In-vitro-Modell des Neuroblastoms unter Verwendung von kollagenbasierten Gerüsten

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.

Brustepithel- und Endothelzell-Sphäroide als potenzielles funktionelles In-vitro-Modell für die Brustkrebsforschung

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.

Quantifizierung der Brustkrebs-Zelle Invasivität Mit einem Drei-dimensionale (3D) Modell

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

Medizin

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.

Verfahren zur Kultivierung humaner Femur Gewebeexplantaten an Brustkrebs Zellbesiedlung des metastasierten Niche Studieren

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.

Dreidimensionale (3D) Tumor Spheroid Invasionsassay

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.

Dreidimensionale Kulturtest auf Krebszelle Invasivität und Satelliten Tumorbildung erkunden

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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