Ce protocole a été testé et optimisé initialement dans le laboratoire du Dr Richard Hynes (MIT). Le financement est assuré par le prix IDEA NYSTEM (à LX) et Ruth L. Kirschstein Prix national Research Service (à LX).

1. Préparation des échantillons <ol><li> Cultiver des cellules confluentes à ~ 70% dans leurs médias spécifiques avec des facteurs de croissance ou de FBS (par exemple, DMEM avec du FBS à 10% pour le MC-1 cellules). Aspirer des médias de la plaque et laver délicatement à plusieurs reprises avec 1 x PBS (8 g / L de NaCl, 0,2 g / L de KCl, 1,15 g / L de Na 2 HPO 4 .7 H 2 O, 0,2 g / L de KH 2 PO 4, pH 7,3). </li><li> Aspirer PBS et ajouter 2 mL de trypsine à 0,05% en versène (0,014 g / L de rouge de phénol et 0,2 g / L d&#39;EDTA-Na dans 1 x PBS, pH 7,2). Secouez doucement la plaque pour faciliter le détachement des cellules de la plaque. Observez les cellules sous un microscope. Il prend généralement 2-5 minutes pour les cellules à se détacher. </li><li> Ajouter un volume suffisant de médias contenant FBS (ou inhibiteur de la trypsine de soja) pour étancher l&#39;activité de la trypsine et de recueillir les cellules dans un tube de 50 ml faucon. Compter les cellules en utilisant un hématimètre. Charge 10 pi de suspension cellulaire sur un hématimètre propre. Le nombre de cellules par mL est égal à la moyenne # des cellules dans chacune des cinq carrés multipliée par 10 4. </li><li> Centrifuger les cellules à 1000 RPM (ou ~ 200 xg) dans une centrifugeuse de paillasse pendant 3 minutes. </li><li> Retirez délicatement les médias, sans perturber le culot cellulaire. Resuspendre les cellules dans un volume approprié de solution tampon Hanks Balanced (HBSS) pour atteindre une concentration finale de ~ 5 x 10 6 cellules / ml. </li><li> Filtrer à travers les cellules d&#39;un Falcon 70 tamis cellulaire um d&#39;exclure des agrégats à grandes cellules. Placez l&#39;extrémité de la pipette directement sur le filtre sur un tube étiqueté culture 5mL Falcon. Vite éjecter la suspension à travers le filtre dans le tube de culture. </li><li> Compter les cellules de nouveau en utilisant un hématimètre et les diluer à une concentration finale de 2,5 x 10 6 / mL. Gardez les cellules sur la glace. </li><li> Déterminer la viabilité des cellules. Mélanger quelques cellules au bleu trypan et de mesurer le pourcentage de morts (en bleu) des cellules sur les cellules total en utilisant un hématimètre. La viabilité des cellules doit être ≥ 90% avant l&#39;injection. </li></ol> 2. Injection par voie intraveineuse <ol><li> Changer de gants. Doucement attraper la queue d&#39;une souris immunodéficientes (nude, NOD-SCID, ou GFN, Laboratoire Jackson) et tirez dans le dispositif de retenue de la souris, avec son dos contre la fente et sa queue qui sortait de la petite ouverture dans le dos de la de contention . </li><li> Faites glisser lentement vers l&#39;intérieur de l&#39;anneau le long de la fente et le verrouiller en place une fois l&#39;anneau des prises de l&#39;embouchure de la souris. La souris ne doit pas être en mesure de se déplacer librement, mais doivent avoir le taux normal de la respiration. </li><li> Trouvez les veines de la queue majeur. Quatre vaisseaux sanguins principaux sont présents dans une queue de souris. Les vaisseaux sanguins sur les côtés dorsale et ventrale de la queue sont les artères. Les veines sont sur les côtés latéraux de la queue. </li><li> Attirer plus de 200 ul de cellules préparées dans une seringue 1ml. Fixez l&#39;aiguille 30 G 1 / 2 pouce et pousser les bulles d&#39;air qui peuvent exister. Le volume final de cellules dans la seringue doit être 200 uL (soit 5 x 10 5 cellules au total). </li><li> Injecter des cellules dans la veine de la queue. <ol class="lalpha"><li> Démarrer à partir de l&#39;extrémité distale de la queue, donc si le premier essai échoue, une région plus proximale de la queue pourrait être utilisé pour un deuxième essai. </li><li> Essuyez la queue avec 70% d&#39;éthanol. Tirez sur la queue droite. Maintenez la pointe de la queue avec le pouce et l&#39;appui du point d&#39;injection avec l&#39;index. </li><li> Insérez l&#39;aiguille de la veine et injecter les cellules. Assurez-vous que l&#39;aiguille et la seringue sont parallèles à la veine lors de l&#39;injection, sinon l&#39;aiguille se pointer à travers la paroi du vaisseau et d&#39;injecter les cellules dans les tissus adjacents queue. </li><li> Retirez l&#39;aiguille après l&#39;injection. Le sang doit abondante à partir du site d&#39;injection si l&#39;injection est allé avec succès. Appuyez sur un morceau propre de serviette en papier ou un coton tige sur le site d&#39;injection pour faciliter la coagulation, et palper la queue vers le haut pour pousser tout échantillon résiduel dans la veine en circulation. </li><li> Relâchez la souris à partir du dispositif de retenue et de le retourner à la cage. Enregistrez le processus d&#39;injection (par exemple, combien d&#39;essais il a fallu à injecter les cellules et combien cellules ont été injectées) dans un cahier de laboratoire. </li><li> Déterminer la viabilité des cellules après l&#39;injection, comme décrit dans la Section de préparation des échantillons, étape 9. Cette étape permet de rassurer que les cellules rester en vie tout au long du processus d&#39;injection. A la fin d&#39;une expérience d&#39;injection, la viabilité des cellules vont diminuer, mais devrait être supérieure à 80%. </li><li> (Optionnel) Isoler les cellules et rincer les restes les boulettes avec du PBS fois. Congelez les boulettes à -80 oC pour les analyses futures (par exemple, Western blot pour confirmer l&#39;expression des gènes ou des passes rabattues). </li></ol></li><li> Généralement, après un ou deux mois, les souris seront disséqués et les emplacements des métastases sont grossièrement déterminée. Du poumon est le site principal de métastases, car il contient le premier lit capillaire que les cellules après leur rencontrepénétrer dans la circulation. </li><li> Après rinçage avec du PBS, chaque lobe du poumon (ou d&#39;autres tissus contenant des métastases) est observé sous un microscope à dissection (figure 1). Le nombre de métastases décelables sur les deux côtés du poumon est compté et le nombre de métastases pulmonaires sur l&#39;ensemble des quatre lobes sont ajoutées comme le nombre total de métastases pulmonaires. 1 métastases pulmonaires sont plus facilement détectées si les poumons sont fixés dans le formol durant la nuit , puisque les métastases se apppear que taches blanchâtres, contrairement à l&#39;sombres adjacents tissus pulmonaires brun. </li></ol> 3. La culture de cellules de métastases pulmonaires <ol><li> Métastases pulmonaires sont isolés de souris injectées. Chacun doit être cultivées séparément. </li><li> Chaque métastase est hachée par la fin d&#39;un capuchon de l&#39;aiguille (stérile) sur un tamis 70 pm cellule. </li><li> Rincer la crépine de cellule avec plusieurs mls de milieu et les cellules recueillies qui passent par le filtre dans une boîte de culture. </li><li> Incuber les cellules à 37 ° C pendant au moins quatre jours sans perturbation. </li><li> Laver le sang ou les débris de tissu sur le plat avec du PBS. </li><li> Ajouter un milieu frais. Au début, les cellules cancéreuses et les cellules fibroblastes se développent sur les plaques, mais progressivement, les cellules de fibroblastes va mourir et être remplacées par des cellules cancéreuses. Si les cellules cancéreuses portent pas de gènes résistant aux médicaments, sélectionnez les cellules avec des antibiotiques correspondants. </li><li> La pureté des cellules dérivées est évaluée par immunomarquage utilisant des anticorps contre des protéines spécifiques de l&#39;homme. Nous utilisons un anticorps anti-vimentine humaine (NCL-VIM-V9, Novocastra). Les cellules nous avons dérivé contiennent typiquement plus de 99% de cellules humaines, même en l&#39;absence de sélection des médicaments. Les cellules nouvellement obtenues pourraient être injectés dans des souris immunodéficientes nouveau de tester leurs capacités métastatiques, comme décrit ci-dessus. </li></ol> 4. Les résultats représentatifs <ol><li> A la fin de cet essai, une ligne du cancer métastatique des cellules hautement donne habituellement lieu à de nombreuses métastases pulmonaires (figure 1), 1 alors que les métastases pulmonaires très peu proviennent d&#39;une lignée de cellules cancéreuses métastatiques mal. </li><li> Les cellules obtenues à l&#39;aide de cette méthode donne généralement lieu à plus de métastases que la lignée parentale, quand ils sont testés à nouveau en utilisant ce dosage. 1-3 </li></ol><img src="/files/ftp_upload/1942/1942fig1.jpg" alt="Figure 1" /> Images représentant la figure 1. Des poumons de souris après des injections dans la veine caudale des cellules cancéreuses. 5 x 10 5 de la ligne de mélanome métastatique de cellules humaines, cellules SM, 4 ont été injectés dans la veine de la queue des souris immunodéficientes. Deux mois plus tard, les poumons ont été isolées et les métastases ont été trouvés dispersés parmi le tissu pulmonaire normal.

<ol>
	<li>Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+an+animal+melanoma+model+correlate+with+aggressiveness+of+human+melanoma+metastases.">Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases.</a> Mol Cancer Res. 6 (5), 760-760 (2008).</li><li>Clark, E. A., Golub, T. R., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC.">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406 (6795), 532-532 (2000).</li><li>Xu, L., Begum, S., Hearn, J. D., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPR56,+an+atypical+G+protein-coupled+receptor,+binds+tissue+transglutaminase,+TG2,+and+inhibits+melanoma+tumor+growth+and+metastasis.">GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.</a> Proc Natl Acad Sci U S A. 103 (24), 9023-9023 (2006).</li><li>Kozlowski, J. M., Hart, I. R., Fidler, I. J., Hanna, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+melanoma+line+heterogeneous+with+respect+to+metastatic+capacity+in+athymic+nude+mice.">A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.</a> J Natl Cancer Inst. 72 (4), 913-913 (1984).</li><li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multigenic+program+mediating+breast+cancer+metastasis+to+bone.">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-537 (2003).</li></ol>

Les métastases sont la principale cause de décès chez les patients atteints de cancer. Elle comporte quatre grandes étapes: le détachement des cellules cancéreuses de leurs loci primaire, leur mise en circulation (intravasation), leur sortie de la circulation (extravasation), et la survie et la croissance dans un organe à distance. Métastase dans l&#39;homme est considéré comme un processus lent et qui se manifeste souvent après des années de latence. Pour étudier sa progression en temps opportun, le test c...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-021</td>
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<td>Hyclone</td>
<td>SH30088.03</td>
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<td>Invitrogen</td>
<td>14175-095</td>
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<td>&nbsp;</td>
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<td>S9888</td>
<td>&nbsp;</td>
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<td>6838-04</td>
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<td>Sigma-Aldrich</td>
<td>P3532</td>
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<td>4010</td>
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<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15250-061</td>
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<td>Invitrogen</td>
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<td>VWR</td>
<td>89039-656</td>
<td>&nbsp;</td>
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<td>Axygen Scientific</td>
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<td>BD Biosciences</td>
<td>329650</td>
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<td>30 &frac12; gauge needle</td>
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<td>BD Biosciences</td>
<td>305106</td>
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<td>Mouse restrainer</td>
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<td>Plas Labs Inc.</td>
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<td>&nbsp;</td>
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<td>08-902</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ1500</td>
<td>&nbsp;</td>
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<td>Benchtop Centrifuge</td>
<td>&nbsp;</td>
<td>Thermoscientific</td>
<td>75003491</td>
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experimental metastasis assay

Les métastases sont la principale cause de décès chez les patients atteints de cancer. Pour comprendre le mécanisme de métastases, un essai de métastases expérimentales a été établi en utilisant des souris immunodéficientes. Cet article définit les procédures impliquées dans cet essai, y compris la préparation des échantillons, l&#39;injection intraveineuse, et la culture de cellules de métastases pulmonaires. Brièvement, un nombre prédéterminé de cellules cancéreuses humaines ont été préparés<em&gt; In vitro</em&gt; Et directement injectées dans la circulation des souris immunodéficientes dans leurs veines de la queue. Un petit nombre de cellules à survivre aux turbulences dans la circulation et de grandir comme des métastases dans les organes internes, comme les poumons. Les souris injectées sont disséqués après une certaine période. La distribution tissulaire des métastases est déterminé sous un microscope à dissection. Le nombre de métastases dans un tissu spécifique est compté et il est directement corrélée avec la capacité métastatique des cellules cancéreuses injectées. Les métastases sont apparues isolées et cultivées<em&gt; In vitro</em&gt; Que les lignées cellulaires, qui présentent souvent des capacités accrues métastatique à la lignée parentale lorsqu&#39;il est injecté à nouveau dans des souris immunodéficientes. Ces dérivés hautement métastatiques deviennent des outils utiles pour identifier les gènes ou les voies moléculaires qui régulent la progression métastatique.

Watch this Scientific Journal Video about Experimental Metastasis Assay at JoVE.com

Experimental Metastasis Assay

Cet article décrit les procédures d&#39;un essai de métastases expérimentales qui est utilisé pour déterminer le potentiel métastatique de lignées cellulaires humaines de cancer.

Assay Métastases expérimentales

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Research

JoVE Journal

Medicine

20.2K vues.  University of Rochester Medical Center. Cet article décrit les procédures d'un essai de métastases expérimentales qui est utilisé pour déterminer le potentiel métastatique de lignées cellulaires humaines de cancer.

Vidéo: Experimental Metastasis Assay

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

Analyse quantitative des métastases du cancer en utilisant un modèle d&#39;embryon aviaire

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

Recherche

Médecine

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Test in vitro avec dégagement mésothéliales que les modèles les étapes précoces de métastases du cancer de l&#39;ovaire

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Modèles de métastases osseuses

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Expérimentale transport pneumocoque humaines

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Le<em&gt; En ovo</em&gt; CAM-test comme un Xenograftmodel pour le sarcome

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un modèle murin orthotopique de cancer de la prostate humain métastases

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Tumeur primaire et MEF cellule d&#39;isolement pour étudier métastases pulmonaires

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

Chick Coeur Invasion Assay pour tester les techniques invasives des cellules cancéreuses et l&#39;activité des composés potentiellement anti-invasives

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Un «Patient-Like&quot; Orthotopique Syngénique modèle de souris de carcinome hépatocellulaire Métastases

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Quantification du commerce intra-péritonéale Cancer de l&#39;ovaire Métastase

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...