Nous remercions sincèrement le Dr Chen Wenjin pour son aide avec le programme MATLAB et l&#39;Institut du Cancer du New Jersey histopathologie installation et installation de microscopie. Nous remercions également les relecteurs pour leurs commentaires et suggestions. Cette étude est soutenue par le Raymond et Beverly Sackler Fondation pour la recherche.

<ol>
	<li>Taal, B. G., Visser, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+neuroendocrine+tumours.">Epidemiology of neuroendocrine tumours.</a> Neuroendocrinology. 80, 3-7 (2004).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat. Methods. 4, 359-365 (2007).</li><li>Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid-based+drug+screen:+considerations+and+practical+approach.">Spheroid-based drug screen: considerations and practical approach.</a> Nat. Protocol. 4, 309-324 (2009).</li><li>Chan, T. F., Vese, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+contours+without+edges.">Active contours without edges.</a> IEEE Trans Image Process. 10, 266-277 (2001).</li><li>Olert, J., Wiedorn, K. H., Goldmann, T., K&#252;hl, H., Mehraein, Y., Scherthan, H., Niketeghad, F., Vollmer, E., M&#252;ller, A. M., M&#252;ller-Navia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HOPE++Fixation:+A+Novel+Fixing+Method+and+Paraffin-embedding+Technique+for+Human+Soft+Tissues.">HOPE Fixation: A Novel Fixing Method and Paraffin-embedding Technique for Human Soft Tissues.</a> Pathology-Research and Practice. 197, 823-826 (2001).</li><li>Baradari, V., Huether, A., H&#246;pfner, M., Schuppan, D., Scher&#252;bl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiproliferative+and+proapoptotic+effects+of+histone+deacetylase+inhibitors+on+gastrointestinal+neuroendocrine+tumor+cells.">Antiproliferative and proapoptotic effects of histone deacetylase inhibitors on gastrointestinal neuroendocrine tumor cells.</a> Endocr. Relat. Cancer. 13, 1237-1250 (2006).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Hanging+Drop+Cell+Culture+Protocol+for+Generation+of+3D+Spheroids.">A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids.</a> J. Vis. Exp. (51), e2720 (2011).</li><li>Shaw, K. R., Wrobel, C. N., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+three-dimensional+basement+membrane+cultures+to+model+oncogene-induced+changes+in+mammary+epithelial+morphogenesis.">Use of three-dimensional basement membrane cultures to model oncogene-induced changes in mammary epithelial morphogenesis.</a> J. Mammary Gland Biol. Neoplasia. 9, 297-310 (2004).</li><li>Ivascu, A., Kubbies, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+single-tumor+spheroids+for+high-throughput+cell+function+and+toxicity+analysis.">Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.</a> J. Biomol. Screen. 11, 922-932 (2006).</li><li>Napolitano, A. P., Chai, P., Dean, D. M., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+Self-Assembly+of+Complex+Cellular+Aggregates+on+Micromolded+Nonadhesive+Hydrogels.">Dynamics of the Self-Assembly of Complex Cellular Aggregates on Micromolded Nonadhesive Hydrogels.</a> Tissue Engineering. 13, 2087-2094 (2007).</li><li>do Amaral, J. B., Rezende-Teixeira, P., Freitas, V. M., Machado-Santelli, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF-7+cells+as+a+three-dimensional+model+for+the+study+of+human+breast+cancer.">MCF-7 cells as a three-dimensional model for the study of human breast cancer.</a> Tissue Eng. Part C Methods. 17, 1097-1107 (2011).</li><li>Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+An+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: An underestimated tool is catching up again.</a> Journal of Biotechnology. 148, 3-15 (2010).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Nous avons démontré une méthode simple, fiable et reproductible pour former 3D sphéroïde multicellulaire à partir de cellules de tumeurs neuroendocrines utilisant agarose recouvertes de plaques à 24 puits. Le succès de cette technique est la possibilité d&#39;obtenir un sphéroïde unique avec la taille et la forme homogène uniforme par jour 6 à partir d&#39;un nombre fixe de cellules. L&#39;étape clé est l&#39;agitation lente de la plaque avec des cellules NET pour la première 16-24 heures après placage. Cette méthode peut également s&#39;appliquer à commune lignée cellulaire humaine de cancer du sein MCF-7, lignée cellulaire humaine du côlon adénocarcinome HT-29, l&#39;homme non à petites cellules du poumon carcinome ligne H1299 et humaines lignée de cellules embryonnaires de rein HEK-293. Ils peuvent tous former uniformes et compact sphéroïdes 3D pour écran thérapeutique (données non présentées). Bien que plusieurs méthodes sont signalés sur la génération de sphéroïdes 3D, 7-10 la méthode décrite ici ne nécessite pas de plaques spécialisées ou des réactifs et est donc simple et rentable. Notre demonstrat données inéditese que NET sphéroïdes 3D cultivées sur agarose d&#39;imiter les xénogreffe tumorale in vivo NET géométriquement et moléculaire, par rapport aux cellules monocouches 2D. Ceci est similaire à ce qui a été rapporté pour les sphéroïdes 3D réalisées à partir sur d&#39;autres lignées cellulaires cancéreuses humaines. 11,12 La limitation de la modèle de culture cellulaire 3D, c&#39;est qu&#39;il ne peut pas remplacer complètement tester l&#39;efficacité in vivo de drogues ou de traitements thérapeutiques en raison les sphéroïdes 3D sont comme des tumeurs avasculaires, qui est différente de la dans la tumeur in vivo dans cet aspect. Néanmoins, cette méthode aidera combler le fossé et les évaluations des médicaments thérapeutiques à partir NET in vitro à in vivo dans certains aspects. Immunofluorescence sur sphéroïdes multicellulaires est très difficile à réaliser en raison de la grande taille et la géométrie des sphéroïdes flottants en 3D et la limitation du microscope confocal. Ici, nous illustrons une méthode de traitement à la paraffine plongement HistoGelintégration, permettant la coloration immunohistochimique des coupes sériées en coupe de la sphéroïdes 3D. Par ailleurs, notre coutume développée programme MATLAB permet précise et reproductible estimer la taille des sphéroïdes, de surveiller l&#39;efficacité du médicament de manière efficace, et pourrait être intégrée dans un écran à haut débit dans le développement de médicaments pour les patients NET.

<table border="1"><tbody><tr><td> Nom </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> DMEM/F12 médias </td><td> Gibco </td><td> 10565-018 </td><td></td></tr><tr><td> TrypLE </td><td> Gibco </td><td> 12604 </td><td></td></tr><tr><td> Plaques à 24 puits </td><td> Faucon </td><td> 353047 </td><td></td></tr><tr><td> 70 tamis cellulaire mM </td><td> Fisher Scientific </td><td> 22363548 </td><td></td></tr><tr><td> Bande d&#39;étanchéité stérile </td><td> Nunc </td><td> 236366 </td><td></td></tr><tr><td> Un trichostatine </td><td> Sigma </td><td> T8552 </td><td></td></tr><tr><td> Orbital Shaker </td><td> Fisher Scientific </td><td> 11-402-12 </td><td></td></tr></tbody></table>

human neuroendocrine tumor cell lines as a three-dimensional model for the study of human neuroendocrine tumor therapy

Les tumeurs neuroendocrines (TNE) sont des tumeurs rares, avec une incidence de deux par 100 000 personnes par an, et ils représentent 0,5% de l&#39;ensemble des tumeurs malignes humaines. 1 Autre que la chirurgie pour la minorité des patients qui présentent une maladie localisée, il est bénéfice de survie peu ou pas de la thérapie systémique. Par conséquent, il ya un grand besoin de mieux comprendre la biologie des filets, et en particulier de définir de nouvelles cibles thérapeutiques pour les patients atteints non résécables ou métastatiques des tumeurs neuroendocrines. Culture cellulaire 3D devient une méthode populaire pour le dépistage des drogues en raison de sa pertinence dans la modélisation de la dans l&#39;organisation des tissus in vivo de la tumeur et microenvironnement. 2,3 Les sphéroïdes multicellulaires 3D pourrait fournir de précieuses informations d&#39;une manière plus rapide et moins cher que de passer directement de la 2D Les expériences de culture cellulaire pour la santé animale (murin) modèles. Afin de faciliter la découverte de nouvelles thérapies pour le NET patients, nous avons développé un test in vitro 3D sphéroïdes multicellulaires modèle en utilisant les lignes cellulaires humaines NET. Les cellules sont étalées NET dans un non-adhésif revêtu d&#39;agarose de 24 puits de plaque et incubés dans des conditions physiologiques (5% de CO 2, 37 ° C) avec une agitation très lente des 16-24 ans h après le placage. Les cellules forment sphéroïdes multicellulaires à compter du jour 3 e ou 4 e. Les sphéroïdes devenir plus sphérique par le 6 e jour, à quel point les traitements médicamenteux sont initiées. L&#39;efficacité des traitements pharmacologiques sur les sphéroïdes NET est surveillée sur la base de la morphologie, la forme et la taille des sphéroïdes avec un microscope optique à contraste de phase. La taille des sphéroïdes est estimé automatiquement à l&#39;aide d&#39;un programme personnalisé développé MATLAB basé sur un algorithme de contour actif. En outre, nous démontrons une méthode simple pour traiter le plongement HistoGel sur ces sphéroïdes 3D, permettant l&#39;utilisation de standards techniques histologiques et immunohistochimiques. <pclass = &quot;jove_content&quot;&gt; C&#39;est le premier rapport sur la production de sphéroïdes 3D utilisant des lignées cellulaires NET pour examiner l&#39;effet de médicaments thérapeutiques. Nous avons également procédé, l&#39;histologie sur ces sphéroïdes 3D, et a présenté un exemple d&#39;effet d&#39;un seul médicament sur la croissance et la prolifération des sphéroïdes NET. Nos résultats confirment que les sphéroïdes NET sont précieux pour les études de la biologie NET et le développement de médicaments.

Watch this Scientific Journal Video about Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy at JoVE.com

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Nous présentons une simple superposition d&#39;agarose plate-forme pour développer 3D sphéroïdes multicellulaires en utilisant des lignées cellulaires cancéreuses neuroendocrines. Cette méthode fournit un moyen très pratique pour examiner l&#39;effet des médicaments thérapeutiques sur les cellules de tumeurs neuroendocrines. Il pourrait aussi nous aider à établir l&#39;homme sphéroïdes de tumeurs neuroendocrines pour le traitement du cancer.

L&#39;homme neuroendocriniennes lignées cellulaires tumorales comme un modèle tridimensionnel pour l&#39;étude de la thérapie des tumeurs neuro-endocrines humaines

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

human-neuroendocrine-tumor-cell-lines-as-three-dimensional-model-for

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28.5K vues.  Raymond and Beverly Sackler Foundation. Nous présentons une simple superposition d'agarose plate-forme pour développer 3D sphéroïdes multicellulaires en utilisant des lignées cellulaires cancéreuses neuroendocrines. Cette méthode fournit un moyen très pratique pour examiner l'effet des médicaments thérapeutiques sur les cellules de tumeurs neuroendocrines. Il pourrait aussi nous aider à établir l'homme sphéroïdes de tumeurs neuroendocrines pour le traitement du cancer.

Vidéo: Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel haute résolution<em&gt; In vivo</em&gt; Technique d&#39;imagerie pour étudier la réponse dynamique des structures intracrâniennes à la croissance tumorale et de thérapeutique

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Recherche

Médecine

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Tissue Culture Modèle tridimensionnel pour étudier primaire moelle osseuse humaine et de ses affections malignes

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

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

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

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

En trois dimensions modèle de co-culture tumeur stromale-Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Évaluation des leucocytes infiltrant les tumeurs sous-ensembles dans un Modèle de tumeur sous-cutanée

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Mise en place d&#39;un modèle de xénogreffe humain de myélome multiple dans le poulet pour étudier la croissance tumorale, l&#39;invasion et l&#39;angiogenèse

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionnelle (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

Dynamique de suivi de la tumeur du poumon pour stéréotaxique ablatif radiothérapie du corps

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Un modèle de culture de coupe organotypique du glioblastome humain pour l&#39;étude de la migration des cellules tumorales et des effets spécifiques des patients contre les médicaments anti-invasifs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...