We are thankful to Dr. Richard Cerione, Dr. Marc Antonyak, and Kelly Sullivan, Cornell University, for providing technical advice, and to Dr. Gerlinde Van de Walle, Cornell University, for sharing their Olympus CKX41 inverted microscope.

1. Préparation de 3% de 2-hydroxyéthyle Agarose <ol><li> Dans un endroit propre, sec 100 ml bouteille en verre, ajouter 0,9 g de 2-hydroxyethyl agarose (Agarose VII), suivie par 30 ml d&#39;eau distillée. </li><li> Micro-ondes le mélange pendant 15 secondes et agiter doucement. Répétez cette étape au moins trois fois jusqu&#39;à ce que la poudre d&#39;agarose dissout complètement. </li><li> Autoclaver la bouteille contenant la solution pendant 15 min-. </li><li> Laisser la solution d&#39;agarose à refroidir à température ambiante avant une nouvelle utilisation. Conserver la solution à la température ambiante. </li></ol> 2. Préparation de la couche inférieure: 0,6% gel d&#39;agarose <ol><li> Pré-réchauffer plusieurs 5 ml et 10 ml de pipettes dans un incubateur à 37 ° C pour empêcher la solidification de l&#39;agarose dans la pipette lors de la manipulation. </li><li> Desserrer partiellement le couvercle de la bouteille et micro-ondes la solution d&#39;agarose à pré-mesure de 3% de 2-hydroxyéthyle pendant 15 s. Ensuite, mélanger doucement la solution et micro-ondes pendant 15 secondes. ATTENTION: Soyez prudent lorsque vous tourner le agarosolution soi parce que la solution se lève lorsqu&#39;il est exposé à l&#39;air et peut déborder. </li><li> Si il ya gel solide résiduel dans la bouteille, micro-ondes quelques secondes de plus. </li><li> Gardez la bouteille contenant la solution d&#39;agarose dans un bain d&#39;eau à 45 ° au cours des prochaines étapes pour empêcher la solution d&#39;agarose de se solidifier prématurément. </li><li> Médias MCF10DCIS chaud dans un bain d&#39;eau à 37 C ° de. Remarque: MCF10DCIS support est constitué de DMEM / F12, de sérum de cheval à 5%, 5% de pénicilline streptomycine. </li><li> Transfert 3 ml de la solution d&#39;agarose à 3% en utilisant les pipettes préchauffées dans un tube conique de 50 ml stérile. </li><li> Immédiatement ajouter 12 ml de milieu de MCF10DCIS chaudes et inverser doucement le tube conique de mélanger l&#39;agarose avec les médias. Essayez de ne pas former des bulles comme il va interférer avec la colonie comptant plus tard. </li><li> Ajouter doucement 2 ml de ce mélange dans chaque puits d&#39;une plaque de culture à 6 puits sans former de bulles d&#39;air. </li><li> Incuber le 6 puits plaque de culture horizontally sur une surface plane à 4 ° C pendant 1 heure pour permettre au mélange de se solidifier. </li><li> Après le mélange se solidifie, placer la plaque dans un incubateur à 37 ° de 30 min. La couche inférieure est maintenant prêt à l&#39;emploi. </li></ol> 3. Préparation de la couche contenant des cellules: 0,3% gel d&#39;agarose <ol><li> Trypsiniser les cellules MCF10DCIS et les diluer à une concentration cellulaire de 4 x 10 4 / ml. </li><li> Prendre 2 ml de l&#39;agarose à 3% en utilisant des pipettes préchauffées et transférer dans un tube conique de 50 ml stérile. </li><li> Immédiatement ajouter 8 ml de MCF10DCIS médias pour le tube conique et inverser doucement pour mélanger l&#39;agarose avec les médias. Éviter la formation de bulles. </li><li> Prendre 2 ml de cellules MCF10DCIS (4 x 10 4 / ml) et traiter avec BB-CLA (0 uM (DMSO) ou 1 um). </li><li> Dans une dilution 1: 1, le mélange des cellules avec le 0,6% d&#39;agarose. </li><li> Prélever 1 ml du mélange de cellules-agarose et ajouter doucement sur la couche inférieure de la culture à 6 puits pfin (2 x 10 4 cellules / ml). </li><li> Placer la plaque de culture à 6 puits à l&#39;horizontale sur une surface plane à 4 ° C pendant au moins 15 minutes pour permettre à la couche de recouvrement se solidifier. </li><li> Après le mélange se solidifie, placer la plaque dans un incubateur à 37 ° C pendant de une semaine avant d&#39;ajouter la couche d&#39;alimentation. </li></ol> 4. Préparation de la couche d&#39;alimentation: 0,3% Agarose Gel <ol><li> Micro-ondes de la solution d&#39;agarose préfabriqué 3% de 2-hydroxyéthyle pendant 15 s. agiter doucement la solution et micro-ondes pendant 15 secondes. </li><li> Equilibrer la bouteille de solution de gélose dans un bain d&#39;eau à 45 ° C. </li><li> Réchauffez les médias MCF10DCIS dans un bain d&#39;eau à 37 ° C. </li><li> Mélanger 1 ml de solution d&#39;agarose à 3% avec 9 ml de milieu de MCF10DCIS chaud dans un tube conique de 50 ml et inverser doucement pour mélanger l&#39;agarose avec les médias. Éviter la formation de bulles d&#39;air. </li><li> On traite le mélange avec BB-CLA (0 uM (DMSO) ou 1 pM). </li><li> Ajouter délicatement 1 ml de ce mélange (sans pourming bulles) dans chaque puits de la plaque de culture de 6 puits contenant le fond et couches molles. </li><li> Placer la plaque de culture à 6 puits à l&#39;horizontale sur une surface plane à 4 ° C pendant au moins 15 minutes pour permettre au mélange de se solidifier. </li><li> Après la couche d&#39;alimentation se solidifie, placer la plaque dans un incubateur à 37 ° C. </li><li> Répétez cette procédure hebdomadaire alimentation en superposant 1 ml de solution d&#39;agarose / moyen / traitement de 0,3% sur la couche d&#39;alimentation existante pour reconstituer les cellules avec les nouveaux médias jusqu&#39;à ce que la formation de colonies est observée. Remarque: Agar dans les couches molles et d&#39;alimentation est très doux et, par conséquent, les éléments nutritifs ajoutés à partir de la couche d&#39;alimentation seront facilement diffuser dans la couche contenant des cellules d&#39;atteindre les cellules. </li></ol> 5. Collecte de données <ol><li> Au bout de 2,5 semaines de croissance cellulaire dans la gélose molle, compter le nombre de colonies dans chaque puits en utilisant un microscope optique. Pour faciliter la quantification, imprimer une grille sur une transparence et fixer la grille pour laPlaque de 6 puits pour aider à localiser où les cellules sont en cours de comptage. Étant donné que la taille des colonies (telle que quantifiée par le diamètre de chaque colonie) varie prédéfinir une taille de colonie de référence pour déterminer les colonies qui sera reçu. Par exemple, inclure la taille des colonies de 70 um ou plus dans l&#39;analyse de données. </li><li> Stocker les échantillons à 4 ° C pour empêcher la formation de colonies pour le comptage et futur. Sceller le 6 puits plaque de culture avec du Parafilm pour éviter les gels de se dessécher. </li></ol>

<ol>
	<li>Hamburger, A. W., Salmon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+bioassay+of+human+tumor+stem+cells.">Primary bioassay of human tumor stem cells.</a> Science. 197, 461-463 (1977).</li><li>Wang, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signaling+regulating+anchorage-independent+growth+of+cancer+cells.">Molecular signaling regulating anchorage-independent growth of cancer cells.</a> Mt Sinai J Med. 71 (6), 361-367 (2004).</li><li>Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J., Pruijn, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAD,+a+growing+family+of+citrullinating+enzymes:+genes,+features+and+involvement+in+disease.">PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease.</a> Bioessays. 25 (11), 1106-1118 (2003).</li><li>Horibata, S., Coonrod, S. A., Cherrington, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+peptidylarginine+deiminase+enzymes+in+disease+and+female+reproduction.">Role for peptidylarginine deiminase enzymes in disease and female reproduction.</a> J Reprod Dev. 58 (3), 274-282 (2012).</li><li>Mohanan, S., Cherrington, B. D., Horibata, S., McElwee, J. L., Thompson, P. R., Coonrod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+peptidylarginine+deiminase+enzymes+and+protein+citrullination+in+cancer+pathogenesis.">Potential role of peptidylarginine deiminase enzymes and protein citrullination in cancer pathogenesis.</a> Biochem Res Int. , 895343 (2012).</li><li>McElwee, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+PADI2+as+a+potential+breast+cancer+biomarker+and+therapeutic+target.">Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.</a> BMC Cancer. 12, 500 (2012).</li><li>Knight, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidylarginine+deiminase+inhibition+disrupts+NET+formation+and+protects+against+kidney,+skin+and+vascular+disease+in+lupus-prone+MRL/lpr+mice.">Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice.</a> Ann Rheum Dis. , 1-8 (2014).</li><li>Miller, F. R., Santner, S. J., Tait, L., Dawson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF10DCIS.com+xenograft+model+of+human+comedo+ductal+carcinoma+in+situ.">MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ.</a> J Natl cancer Inst. 92, 1185-1186 (2000).</li><li>Fan, D., Morgan, L. R., Schneider, C., Blank, H., Fan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+evaluation+of+human+tumor+chemosensitivity+in+the+soft-agar+assay+and+its+clinical+correlations.">Cooperative evaluation of human tumor chemosensitivity in the soft-agar assay and its clinical correlations.</a> J Cancer Res Clin Oncol. 109, 23-28 (2000).</li><li>Hamburger, A. W., White, C. P., Dunn, F. E., Citron, M. L., Hummel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+human+tumor+colony+growth+in+soft+agar+by+serum.">Modulation of human tumor colony growth in soft agar by serum.</a> Int J Cell Cloning. 1 (4), 216-229 (1983).</li><li>Anderson, S. N., Towne, D. L., Burns, D. J., Warrior, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+soft+agar+assay+for+identification+of+anticancer+compound.">A high-throughput soft agar assay for identification of anticancer compound.</a> J Biomol Screen. 12, 938-945 (2007).</li></ol>

The authors have nothing to disclose.

Le taux de formation de colonies dans de l&#39;agar mou varie en fonction du type de cellule 9. Par conséquent, le nombre de cellules pour commencer devrait être optimisé et ajusté en conséquence. Une gamme départ suggéré est entre 5 x 10 2 à 1 x 10 4 cellules par puits en utilisant une plaque de 6 puits. En outre, la taille des colonies varie en fonction du taux de croissance de chaque cellule. Par conséquent, un prédéfini une coupure pour la taille des colonies est nécessa...

Both non-transformed (normal) and transformed cells can readily proliferate in a 2D monolayer culture. This form of adherent cell growth is quite dissimilar from that which occurs in vivo where, in the absence of mitogenic stimulation, cells do not often rapidly divide within their microenvironment. The soft agar assay on the other hand is distinct from 2D culture systems because it quantifies tumorigenicity by measuring a cell&#8217;s ability to proliferate and form colonies in suspension within a semi-solid agarose gel1. In this setting, non-transformed cells are unable to rapidly propagate in the absence of anchorage to the extracellular matrix (ECM) and undergo apoptosis, a process known as anoikis. In contrast, cells that have undergone malignant transformation lose their anchorage dependence due to activation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and Rac/Cdc42/PAK. Therefore, these cells are able to grow and form colonies within the semi-solid soft agar matrix2.
A common use of the soft agar assay is to test whether specific compounds, such as PADI inhibitors, are able to suppress tumor growth in vitro. In general, colony count or colony sizes are quantitative read-outs from the assay that can be compared between control and treatment groups to assess differences in cellular tumorigenicity. Therefore, if one finds that colony formation is inversely correlated with increasing drug concentration, then a conclusion could be drawn that the drug is an effective inhibitor of tumorigenicity in vitro. On the other hand, if the drug does not affect colony formation, the drug is either not at the appropriate dosage or it is not an effective tumorigenic inhibitor. Aside from using a soft agar assay to test the anti-tumor effect of a drug, this assay can also be used to probe the relationship between a specific gene and tumorigenesis. For example, the effect of suppressing PADI2 expression on tumorigenicity can be addressed by PADI2-specific siRNA treatment.
PADIs are calcium-dependent enzymes that post-translationally modify proteins by converting positively charged arginine residues into neutrally charged citrulline in a process known as citrullination or deimination3-5. We have recently found that peptidylarginine deiminase 2 (PADI2) may function as a novel breast cancer biomarker and that PADI inhibitors represent candidate therapies for early stage breast cancers6. For example, we have previously demonstrated that a &#8220;pan-PADI&#8221; inhibitor, Cl-amidine, suppresses the proliferation of breast cancer cells using 2D monolayers and that the inhibitor suppressed the growth of 3D tumor spheroids6. In this report, we extend these studies, and highlight the utility of the soft agar assay, by testing the efficacy of a new PADI inhibitor, BB-CLA, in suppressing the growth of MCF10DCIS breast cancer colonies7. We note that we used MCF10DCIS cells for this experiment because they are oncogenic derivatives of non-transformed human MCF10A cells and because they contain high steady state levels of PADI2 protein8. We hypothesize that PADI2 enzymatic activity plays a key role in the tumorigenicity of this cell line and that BB-CLA-mediated inhibition of PADI2 activity will suppress cancer progression.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Zeiss Axiopot</td>
			<td>Carl Zeiss Microscopy</td>
			<td>1021859251</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Lonza BioWhittaker</td>
			<td>12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Donor Equine Serum</td>
			<td>Fisher Scientific</td>
			<td>SH30074.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Hydroxyethylagarose: Type VII, low gelling temperature</td>
			<td>Sigma-Aldrich</td>
			<td>39346-81-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of the soft agar colony formation assay to identify inhibitors of tumorigenicity in breast cancer cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Le dosage de formation de colonies sur gélose molle peut être utilisée pour une large gamme d&#39;applications documentant la tumorigénicité des cellules cancéreuses. Un avantage majeur de cette technique est que la matrice semi-solide favorise sélectivement la croissance des cellules qui peuvent proliférer d&#39;une manière indépendante de l&#39;ancrage. Cette caractéristique est principalement exposée par les cellules cancéreuses, mais pas par les cellules normales. Nous utilisons cette technique principa...

Watch this Scientific Journal Video about Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells at JoVE.com

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

L&#39;utilisation de l&#39;essai de formation souple Agar colonie à identifier des inhibiteurs de Tumorigénicité dans les cellules cancéreuses du sein

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

utilization-soft-agar-colony-formation-assay-to-identify-inhibitors

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JoVE Journal

Medicine

28.2K vues.  Cornell University. Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro. 

Vidéo: Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modale d&#39;imagerie de l&#39;angiogenèse dans un modèle de rat nu des métastases osseuses du cancer du sein par imagerie par résonance magnétique, la tomodensitométrie et volumétrique ultrasons

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Recherche

Médecine

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Vivez l&#39;imagerie des réponses de drogues dans le microenvironnement des tumeurs dans les modèles murins de cancer du sein

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Initiation de carcinome du sein métastatique en ciblant de l&#39;épithélium canalaire avec adénovirus Cre: un modèle de souris transgénique roman du cancer du sein

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profilage de microARN œstrogène réglementés dans les cellules du cancer du sein

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Trois Cultures dimensionnelles: un outil pour étudier l&#39;architecture normale Acinaire vs transformation maligne des cellules mammaires

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Injection Orthotopique des cellules du cancer du sein dans le Fat Pad mammaire de souris pour étudier la croissance de la tumeur.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Une<em&gt; Ex vivo</em&gt; Modèle pour étudier action hormone dans le sein humain

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

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

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Mammosphere Formation dosage du sein humain cancer des tissus et des lignées cellulaires

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

Dans vivo et ex vivo approches pour étudier Cancer de l&#39;ovaire métastatique colonisation de Lactée spot Structures en péritonéale adipeux

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...