Die Autoren bedanken sich bei allen Mitgliedern des Coller-Labors für ihren hilfreichen Input. H.A.C. war der Milton E. Cassel-Stipendiat der Rita Allen Foundation. Wir würdigen NIH/NCI 1 R01 CA221296-01A1, NIH 1 R01 AR070245-01A1, Melanoma Research Alliance Team Science Award, Cancer Research Institute Clinical Laboratory Integration Program Award, das Iris Cantor Women's Health Center/UCLA CTSI NIH Grant UL1TR000124, das Koordinierungskomitee für Krebsforschung der Universität von Kalifornien, den Metabolismus Theme Award der David Geffen School of Medicine, das Clinical Translational Science Institute und das Jonsson Comprehensive Cancer Center, Innovationspreise des Broad Stem Cell Research Center (Rose Hills und Ha Gaba), eine Auszeichnung des UCLA SPORE in Prostatakrebs (National Cancer Institute der National Institutes of Health unter der Preisnummer P50CA092131), ein Innovationspreis des Broad Stem Cell Center, des Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research an der UCLA, das Tumorzellbiologie-Trainingsprogramm (USHHS Ruth L. Kirschstein Institutional National Research Service Award # T32 CA009056), das Dermatologie-T32-Programm an der UCLA AR071307 und das UCLA-Trainingsprogramm für Muskelzellbiologie, Pathophysiologie und Therapeutika T32 5 T32 AF 65972.

Die Autoren haben nichts zu offenbaren.

In dem Experiment in Abbildung 1 führte die gleichzeitige Einführung menschlicher dermaler Fibroblasten mit menschlichen A2058-Melanomzellen zu größeren Tumoren als bei der Einführung der Melanomzellen ohne co-injizierte Fibroblasten. Dieser Unterschied konnte anhand des Tumorvolumens und des Tumorgewichts leicht festgestellt werden. Die Ergebnisse stimmen mit mehreren Berichten überein, dass krebsassoziierte Fibroblasten das Tumorwachstum fördern können<...

Innerhalb der Tumormikroumgebung ist einer der prominentesten Zelltypen der krebsassoziierte Fibroblast (CAF)1. Diese karzinomassoziierten Fibroblasten können eine tumorsuppressive Rolle spielen 2,3. Zum Beispiel sezernieren S100A-exprimierende Fibroblasten Kollagene, die Karzinogene einkapseln und vor Karzinombildung schützenkönnen 4. Darüber hinaus verursacht die Depletion von α-Smooth Muscle Actin (SMA)-positiven Myofibroblasten bei Bauchspeicheldrüsenkrebs eine Immunsuppression und beschleunigt das Fortschreiten von Bauchspeicheldrüsenkrebs2. CAFs können sich auch gemeinsam mit Krebszellen entwickeln und das Fortschreiten des Tumors fördern 5,6,7,8. Fibroblasten können extrazelluläre Matrixproteine synthetisieren und sezernieren, die eine tumorfördernde Umgebung schaffen8. Diese extrazellulären Matrixproteine können eine mechanische Versteifung des Gewebes verursachen, die mit einer Tumorprogressionverbunden ist 9,10. Die abgeschiedene extrazelluläre Matrix kann als physikalische Barriere wirken, die die Immuninfiltration hemmt11. Die Matrixablagerung durch CAFs wurde auch mit der Tumorinvasion in Verbindung gebracht, da gezeigt wurde, dass von CAFs erzeugtes Fibronektin die Tumorinvasion fördert12. CAFs fördern die Angiogenese und rekrutieren immunsuppressive Zellen in die Tumormikroumgebung, indem sie transformierenden Wachstumsfaktor-β (TGF-β), vaskulären endothelialen Wachstumsfaktor (VEGF), Interleukin-6 (IL-6) und CXC-Chemokin-Liganden 12 (CXCL12) sezernieren13,14,15. Aufgrund ihrer zentralen Rolle bei der Förderung des Tumorwachstums sind krebsassoziierte Fibroblasten ein neues Ziel für die Krebstherapie 6,16,17,18.
Das folgende Protokoll beschreibt eine Methode zum Testen, wie Fibroblasten das Wachstum von Tumoren in einem etablierten und weit verbreiteten Mausmodell des Tumorwachstums beeinflussen. Um die Bedeutung von Fibroblasten in der Tumormikroumgebung zu verstehen, wurde das Standardprotokoll für die Einführung von Krebszellen in Mäuse zur Überwachung ihres Wachstums modifiziert, um Fibroblasten mit der Einführung von Krebszellen einzubeziehen. Die Krebszellen können subkutan oder intradermal eingeführt werden. Eine intradermale Einführung würde zu Tumoren führen, die von der Haut selbst ausgehen. Xenotransplantate, bei denen Krebszellen und Fibroblasten gemeinsam in Mäuse injiziert werden, stellen ein wichtiges methodisches Werkzeug dar, um die Rolle von Fibroblasten, Subpopulationen von Fibroblasten und Proteinfaktoren bei der Förderung des Krebswachstums zu analysieren 19,20,21. Ein detailliertes Protokoll für die Co-Injektion von Krebszellen und Fibroblasten in Mäuse wird bereitgestellt. Diese Methode kann verwendet werden, um das Vorhandensein oder Fehlen von Fibroblasten zu vergleichen, um Fibroblasten aus verschiedenen Quellen zu vergleichen20 oder um Fibroblasten mit und ohne Expression spezifischer Proteine19 zu vergleichen. Nachdem die Krebszellen und Fibroblasten eingeführt wurden, kann die Tumorgröße im Laufe der Zeit überwacht werden. Am Ende der Experimente können Tumore seziert und gewogen werden. Durch die Überwachung des Tumorwachstums im Laufe der Zeit kann die Bedeutung verschiedener Faktoren analysiert werden.
Es gibt mögliche alternative Ansätze, um die Rolle von Fibroblasten beim Tumorwachstum zu untersuchen. Als Beispiel gibt es Cre-loxed-basierte Modelle, die einen gewebespezifischen Knockout von Genen mit Treibern ermöglichen, die bevorzugt in Fibroblasten exprimiert werden. Solche Ansätze bieten auch die Möglichkeit, die Rolle spezifischer Gene und Signalwege in Fibroblasten für die Tumorprogression zu untersuchen. Im Vergleich zu Cre-lox-basierten Ansätzen würde das bereitgestellte Protokoll einen deutlich schnelleren Ansatz zur Überwachung der Rolle von Fibroblasten darstellen, da das Tumorwachstum über nur wenige Wochen überwacht würde. Der bereitgestellte Ansatz ist auch deutlich kostengünstiger, da keine Kolonien gentechnisch veränderter Mäuse erzeugt und untergebracht werden müssen. Das bereitgestellte Protokoll kann verwendet werden, um die Wirkung des Knockdowns verschiedener Gene mithilfe von shRNAs schnell zu testen, anstatt Mauskolonien entwickeln zu müssen. Der bereitgestellte Ansatz ist auch flexibler, da er einen Vergleich einer unterschiedlichen Anzahl von Fibroblasten, unterschiedlichen Verhältnissen von Krebszellen und Fibroblasten, einen Knockdown verschiedener Gene und sogar einen Vergleich von Fibroblasten aus verschiedenen Gewebestellen oder Spezies ermöglichen würde. Ein Cre-lox-Ansatz hätte den Vorteil, dass die Fibroblasten in den Mäusen in einem physiologischeren Kontext vorhanden sind.
Das hier berichtete Protokoll wäre wertvoll für Wissenschaftler, die die Auswirkungen von Fibroblasten auf das Tumorwachstum schnell und kostengünstig überwachen wollen. Dieses Protokoll ist besonders wertvoll für Wissenschaftler, die verschiedene Untergruppen von Fibroblasten oder Fibroblasten aus verschiedenen Quellen auf das Tumorwachstum untersuchen. Wenn es wichtig ist, dass die Tumorinitiierung in einem physiologischen Kontext erfolgt, sollten gentechnisch veränderte Mausmodelle in Betracht gezogen werden.
Es gibt mehrere mögliche Ansätze für die Durchführung dieser Experimente. Immunkompetente Mäuse können als Wirte verwendet werden, was die Untersuchung von Fibroblasten-Immunzell-Interaktionen ermöglichen würde. Für immunkompetente Mausmodelle müssen Mauskrebszellen und embryonale Fibroblasten (MEFs) der Maus injiziert werden. Die Verwendung von MEFs ermöglicht es dem Forscher auch, die breite Palette von Knockout-Mausstämmen zu nutzen, um das Vorhandensein oder Fehlen eines interessierenden Gens zu testen. Alternativ können immundefiziente Mäuse verwendet werden, um die Rolle menschlicher Fibroblasten bei der Förderung des Wachstums von Tumoren bei Mäusen zu testen, die von menschlichen Krebszellen stammen. Die Einführung der Krebszellen kann subkutan oder orthotopisch erfolgen. Beim Melanom kann, wie unten beschrieben, das Tumor-Fibroblasten-Gemisch intradermal injiziert werden, um eine orthotope Injektion durchzuführen, die die Stelle in der Haut, an der sich ein Melanom entwickeln würde, genauer simuliert.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>26G Needles</td>
			<td>Fisher Scientific</td>
			<td>14-826-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>Fisher Scientific</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal clipper miniARCO with surgical blade #40</td>
			<td>WAHL Professional</td>
			<td>8787-450A</td>
			<td></td>
		</tr>
		<tr>
			<td>Athymic nude mice (NU/J)</td>
			<td>The Jackson labs</td>
			<td>002019</td>
			<td>These mice are immunocompromised and can be used for experiments in which human cells are introduced. Immunocompetent mice can also be used if mouse cancer cells and fibroblasts will be introduced.</td>
		</tr>
		<tr>
			<td>Cancer cells</td>
			<td>ATCC</td>
			<td>ATCC&#174; CRL-11147&#8482;</td>
			<td>This is the catalog number for a primary human melanoma cell line. Other cancer cell types can also be used.</td>
		</tr>
		<tr>
			<td>Cell Culture Multi Flasks</td>
			<td>Fisher Scientific</td>
			<td>14-826-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for conical tubes capable of reaching 180 x g</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber</td>
			<td>Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Fisher Scientific</td>
			<td>11965-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>MT35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblasts&#160;</td>
			<td>ATCC</td>
			<td>PCS-201-012</td>
			<td>We isolate fibroblasts from skin in our lab. This is a catalog number for an adult primary human dermal fibroblast cell line. MEFs and fibroblasts derived from other sites can also be used.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC 11695-6776-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS USP grade for injection into mice</td>
			<td>Fisher Scientific</td>
			<td>50-751-7476</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 10 ml serological pipet</td>
			<td>Celltreat</td>
			<td>667210B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 5 ml serological pipet</td>
			<td>Celltreat</td>
			<td>229005B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 50 ml centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>28-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringe Filters pore size 0.2 microns</td>
			<td>Fisher Scientific</td>
			<td>09-740-61A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tissue culture-grade Trypsin-EDTA</td>
			<td>Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tissue-culture grade PBS</td>
			<td>Fisher Scientific</td>
			<td>50-751-7476</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterle 25 ml serological pipet</td>
			<td>Celltreat</td>
			<td>667225B</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 100 x 20 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-202</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 150 x 20 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-203</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 60 x 15 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-260</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
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</table>

a mouse model to investigate the role of cancer-associated fibroblasts in tumor growth

Krebsassoziierte Fibroblasten (CAFs) können eine wichtige Rolle beim Tumorwachstum spielen, indem sie eine tumorfördernde Mikroumgebung schaffen. Modelle zur Untersuchung der Rolle von CAFs in der Tumormikroumgebung können hilfreich sein, um die funktionelle Bedeutung von Fibroblasten, Fibroblasten aus verschiedenen Geweben und spezifischen genetischen Faktoren in Fibroblasten zu verstehen. Mausmodelle sind unerlässlich, um die Faktoren zu verstehen, die zum Tumorwachstum und zur Tumorprogression in einem In-vivo-Kontext beitragen. Hier wird ein Protokoll bereitgestellt, bei dem Krebszellen mit Fibroblasten gemischt und in Mäuse eingeführt werden, um Tumore zu entwickeln. Die Tumorgrößen im Zeitverlauf und die endgültigen Tumorgewichte werden bestimmt und zwischen den Gruppen verglichen. Das beschriebene Protokoll kann einen besseren Einblick in die funktionelle Rolle von CAFs beim Tumorwachstum und -fortschreiten geben.

A2058 humane Melanomzellen und primäre humane dermale Fibroblasten wurden unter sterilen Bedingungen kultiviert. Die Zellen wurden gesammelt und dreimal mit PBS gewaschen. Immundefiziente Mäuse (NU/J - Foxn1 nude strain) wurden subkutan an einer Flanke mit 0,25 Millionen A2058 Melanomzellen allein injiziert. Auf der anderen Flanke wurde Mäusen eine Mischung aus 0,25 Millionen A2058-Melanomzellen und 0,75 Millionen Fibroblasten injiziert. Zellen wurden in 12 immundefiziente Mäuse injiz...

Watch this Scientific Journal Video about A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth at JoVE.com

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Ein Protokoll zur Co-Injektion von Krebszellen und Fibroblasten und zur Überwachung des Tumorwachstums im Laufe der Zeit wird bereitgestellt. Dieses Protokoll kann verwendet werden, um die molekularen Grundlagen für die Rolle von Fibroblasten als Regulatoren des Tumorwachstums zu verstehen.

Ein Mausmodell zur Untersuchung der Rolle krebsassoziierter Fibroblasten beim Tumorwachstum

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

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Cancer Research

4.3K Aufrufe.  University of California, Los Angeles. Ein Protokoll zur Co-Injektion von Krebszellen und Fibroblasten und zur Überwachung des Tumorwachstums im Laufe der Zeit wird bereitgestellt. Dieses Protokoll kann verwendet werden, um die molekularen Grundlagen für die Rolle von Fibroblasten als Regulatoren des Tumorwachstums zu verstehen.

Serie: A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

Tumor Engraftment in einem Xenograft-Maus-Modell der menschlichen Mantel-Zell-Lymphom

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Forschung

Krebsforschung

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Eine Methode, um die Auswirkungen der Umweltanreicherung Doppelpunkt Microbiome Biodiversitätskonvention in einem Mausmodell Doppelpunkt Tumor definieren

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphopeptide Bereicherung gepaart mit quantitativen markierungsfreie Massenspektrometrie zu untersuchen, die Phosphoproteome bei Prostatakrebs

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

Digitalen Polymerase-Kettenreaktion-Assay für die genetische Variation bei einem sporadischen familiäre adenomatöse Polyposis Patienten mit dem Chip-in-Rohr-Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

In Vivo Hemmung von MicroRNA zur Verringerung des Tumorwachstums bei Mäusen

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Ein 3D-Spheroid-Modell als physiologischeres System für krebsassoziierte Fibroblasten Differenzierung und Invasion In Vitro-Studien

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Isolierung proximaler Flüssigkeiten zur Untersuchung der Tumormikroumgebung des Pankreas-Adenokarzinoms

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Isolierung primärer krebsseziensässiger Fibroblasten aus einem syngenen murinen Modell von Brustkrebs zur Untersuchung gezielter Nanopartikel

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Ein Ex-vivo-Gehirnschnittmodell zur Untersuchung und Bekämpfung des metastasierenden Tumorwachstums von Brustkrebs im Gehirn

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

Ein dreidimensionales Sphäroidmodell zur Untersuchung der Tumor-Stroma-Wechselwirkung bei hepatozellulären Karzinomen

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...