Ce travail a été soutenu par le NIH AI P01 068 730 (SNC, N.-É.), le NIH CA016520 / TAPITMAT (N.-É.), le financement privé de la Fondation Claneil (N.-É.), et l&#39;ovaire SPORE accorder à la CCCC et la University of Pennsylvania ( P50 CA83638) et le Fox Chase Cancer Center de base de subvention (P30 CA06927) (CDC). Les auteurs remercient l&#39;assistance technique de l&#39;excellente Core Facility / bioluminescence optique dirigée par le Dr EJ Delikatny à l&#39;Université de Pennsylvanie Secreto, Anthony de la cellule souche et Core xénogreffe dirigé par le Dr. G. Danet-Desnoyers à l&#39;Université de Pennsylvanie cancer Centre de formation pour SNC technique d&#39;injection orthotopique et Denada Dangaj à l&#39;Université de Pennsylvanie / OCRC pour aider la chirurgie.

1. Culture de cellules <ol><li> Avant l&#39;injection orthotopique, la culture des cellules MOV1 Kat, dérivées de tumeurs DR26, dans un flacon T175 jusqu&#39;à ce qu&#39;elles soient confluentes à 90%. Régime d&#39;utiliser 1 à 5 millions de cellules par injection, ce qui nécessitera 1 ou 2 flacons T175. </li><li> Le jour de l&#39;injection, la récolte des cellules et de déterminer le nombre de cellules en utilisant un hématimètre. </li><li> Une fois la concentration cellulaire a été déterminé, un culot cellulaire par centrifugation pendant 5 minutes à 300 X g à température ambiante. </li><li> Après le spin, remettre les cellules d&#39;avoir 1 million en 10 microlitres de PBS stérile avec 0,002 M d&#39;EDTA </li></ol> 2. Pré-Chirurgie <ol><li> Avant la chirurgie, remplir une seringue à insuline 3/10cc avec 1 million MOV1-Kat cellules dans 10 microlitres de PBS EDTA. </li><li> Transfert d&#39;une souris isofluorane anesthésiés à un coussin chauffant. Ajouter pommade ophtalmique pour prévenir la déshydratation des yeux. Ensuite, insérez immédiatement la tête de l&#39;animal dans un système de cône de nez relié à un vaporisateur isoflurane pour offrir l&#39;anesthésie tout au long de la chirurgie. </li><li> Après désinfection du site d&#39;injection avec des tampons d&#39;alcool, sous-cutanée injecter 5 mg / kg de kétoprofène, un analgésique pré-opératoire, avec une seringue à insuline 3/10cc. </li><li> Utiliser tondeuses, se raser la partie gauche de la face dorsale caudale de la jonction thoraco-lombaire à la base de la queue des animaux. Appliquez la crème d&#39;épilation pour supprimer complètement les cheveux. Ensuite, enlever l&#39;excédent avec une serviette en papier humide. </li><li> Une fois que les cheveux ont été enlevés, stériliser la zone rasée avec povidone-iode et tampons imbibés d&#39;alcool. Ensuite, placez un champ opératoire autour de la zone d&#39;incision. </li></ol> 3. Chirurgie <ol><li> Juste avant la chirurgie, ajuster les niveaux de vaporisateur isoflurane à 1,5%. </li><li> Vérifiez que l&#39;animal est totalement anesthésié par pincement du coussinet plantaire. </li><li> Ensuite, localisez la rate sous la peau. Ensuite, en utilisant des ciseaux chirurgicaux, faire une incision de 1-2 cm de long dorsolatéral en haut à droite de la rate. </li><li> Disséquer le rétropéritoine. Le tampon entourant l&#39;ovaire de souris seront observées. Utilisez une pince incurvée à saisir et à exposer le coussinet adipeux entourant l&#39;ovaire de souris. </li><li> Hydrater l&#39;orgue avec quelques gouttes de PBS stérile. </li><li> Utilisez une pince incurvée à saisir, se rétractent ... La position ... et puis fixez l&#39;ovaire pour l&#39;injection </li><li> Alors saisissant fermement l&#39;ovaire avec la pince, injecter 10 microlitres de cellules tumorales MOV1 Kat dans l&#39;ovaire. Une solide connaissance permettra d&#39;éviter la régurgitation de liquide ou de fuite. </li><li> Immédiatement après l&#39;injection, relâchez la tension exercée par la pince. La perforation de l&#39;ovaire devrait spontanément se rétractent et se fermer. </li><li> En utilisant une suture absorbable acide glycolique attaché à une aiguille, fermer la plaie rétro-péritoine. </li><li> Communiqué de l&#39;animal du cône de nez. </li><li> Étirer la peau et sceller les bords de la plaie dorsolatéral avec quelques gouttes de colle tissulaire. </li><li> Enfin, par voie orale d&#39;administrer 100 microlitres d&#39;antibiotiques pour les animaux. Puis le replacer dans sa cage et à surveiller pour la récupération. Gardez l&#39;animal sur les antibiotiques dans l&#39;eau potable pendant une semaine. </li></ol> 4. Imagerie in vivo <ol><li> Une semaine après l&#39;injection orthotopique du MOV1 cellules tumorales Kat, effectuer l&#39;imagerie in vivo. Commencez par transférer une souris isofluorane anesthésiés à la chambre d&#39;imagerie. </li><li> Tournez le niveau vaporisateur isoflurane à 2%. </li><li> Effectuer l&#39;imagerie in vivo selon les instructions du fabricant du système d&#39;imagerie de. Dans cette vidéo, le système de Lumina sera utilisé. </li><li> Pour l&#39;image, cliquez sur l&#39;image vivante du logiciel de bureau icône. Puis, sur le panneau de contrôle IVIS Acquisition, sélectionnez &quot;Initialiser&quot;. Les réglages de l&#39;instrument sont analogues à un réglages de l&#39;appareil </li><li> Sur le panneau de contrôle d&#39;acquisition mis en place les paramètres d&#39;acquisition de l&#39;instrument. Pour la fluorescence, cochez la case &quot;Fluorescent&quot;. Cliquez sur la case Photographie d&#39;acquérir une photographie avec chaque image. </li><li> Ensuite, réglez le temps d&#39;exposition automatique. Sous la rubrique «pixel binning ou CCD résolution&quot;, cochez &quot;Medium&quot;. Puis, sous F / arrêt ou l&#39;ouverture, vérifier la valeur de 2. Ensuite, sélectionnez le filtre d&#39;excitation 535 et le filtre d&#39;émission DsRed. </li><li> Sous le champ de vision, cliquez sur Afficher l&#39;image B à une seule souris. </li><li> Ensuite, cliquez sur l&#39;acquisition de commencer l&#39;acquisition des images. </li><li> Une fois l&#39;acquisition de l&#39;image est terminée, utilisez la région d&#39;intérêt, ou ROI, outil de mesure du signal. Cliquez sur l&#39;icône de mesure de libérer les valeurs de signal et de la zone. </li><li> Enfin, cliquez sur «Enregistrer» pour enregistrer l&#39;image dans le dossier utilisateur </li><li> Après les images ont été sauvegardées, arrêter l&#39;administration de l&#39;isoflurane et le retour de la souris dans sa cage. La souris doit se réveiller immédiatement. </li></ol> 5. Les résultats représentatifs -En utilisant ce protocole, La croissance in vivo d&#39;un cancer de l&#39;ovaire orthotopique peuvent être surveillés pendant au moins 3 semaines à l&#39;aide d&#39;une procédure non-invasive. <img src="/files/ftp_upload/2146/2146fig1.jpg" alt="Figure 1" /> Figure 1. MOV1 cellules Kat, ou PBS comme témoin négatif, ont été injectés dans orthotopique l&#39;ovaire de la non-tumorale des souris sujettes (animal à droite et à gauche et animales, respectivement). Imagerie in vivo a été réalisée deux semaines plus tard. L&#39;émission de fluorescence produite par les cellules greffées Kat MOV1 dans l&#39;ovaire a été mesurée et comparée à celle de souris témoins négatifs. <img src="/files/ftp_upload/2146/2146fig2.jpg" alt="Figure 2" /> Figure 2. Coupes congelées des tumeurs ovariennes MOV1 Kat ont été colorés avec biotinylé anti-CD4 mAb suivie substrat DAB (brun foncé) pour détecter les lymphocytes infiltrant la tumeur (flèche noire). Les cellules ont été contre-colorées avec de méthyle-vert de visualiser les noyaux cellulaires (bleu). Les cellules tumorales (flèches rouges) sont morphologiquement distincts des cellules T. La diapositive apparaît agrandie 40x.

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La chirurgie et les injections orthotopique D&#39;injection orthotopique à Bursa ovaire exigences de formation et de précision. Ainsi <ol><li> En cas de mauvaise expérience, la pratique chirurgicale avec des cadavres d&#39;abord. </li><li> Utilisez de préférence des femelles multipares (une ou deux portées) car ils développent plus ovaires au cours du temps ce qui facilite l&#39;injection et d&#39;augmenter la survie à comparer avec des femelles nullipares. </li><li> En raison de l...

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM-GLUTAMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10564-011</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14040</td>
<td>&nbsp;</td>
<tr>
<td>Versene</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-711E</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Deltaphase</td>
<td>39 DP</td>
<td>&nbsp;</td>
<tr>
<td>Povidone pads</td>
<td>&nbsp;</td>
<td>Dynarex</td>
<td>1108</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>06-669-62</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tears ointment </td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>17845-153</td>
<td>&nbsp;</td>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Fort Dodge laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3cc/insulin syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309301</td>
<td>&nbsp;</td>
<tr>
<td>Polyg Polyglycolic Acid suture/needle (3/8 19mm)</td>
<td>&nbsp;</td>
<td>Syneture</td>
<td>9612-31</td>
<td>&nbsp;</td>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>Vetbond</td>
<td>3M</td>
<td>&nbsp;</td>
<tr>
<td>Vet Bactrim/ oral suspension</td>
<td>&nbsp;</td>
<td>Hi-tech Pharmacal</td>
<td>840823</td>
<td>&nbsp;</td>
<tr>
<td>IVIS-Lumina</td>
<td>&nbsp;</td>
<td>Caliper lifesciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isofluorane</td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>J108013</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum, Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437036</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>TurboFP635 mammalian vector</td>
<td>&nbsp;</td>
<td>Evrogen</td>
<td>FP721</td>
<td>&nbsp;</td>
<tr>
<td>T175 flasks</td>
<td>&nbsp;</td>
<td>cellstar</td>
<td>660-190</td>
<td>&nbsp;</td>		</tbody>
		</table>
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an orthotopic model of serous ovarian cancer in immunocompetent mice for in vivo tumor imaging and monitoring of tumor immune responses

Contexte: Le cancer de l&#39;ovaire est généralement diagnostiqué à un stade avancé où le ratio cas / décès est élevé et reste donc le plus meurtrier de tous les cancers gynécologiques parmi les femmes américaines 1,2,3. Tumeurs séreuses sont les formes les plus répandues de cancer des ovaires et de l&#39;4,5 transgéniques Tg-MISIIR-TAG représente le modèle de souris seulement que se développe spontanément ce type de tumeurs. Tg-MISIIR-Tag souris expriment SV40 région transformer sous le contrôle des substances inhibitrices de Müller récepteur de type II (MISIIR) 6 promoteur du gène. D&#39;autres lignées transgéniques ont été identifiés qui expriment le transgène TAg SV40, mais ne développent pas de tumeurs de l&#39;ovaire. Non-tumorale souris sujettes présentent durée de vie typique des souris C57Bl / 6 et sont fertiles. Ces souris peuvent être utilisés comme receveurs d&#39;une allogreffe de cellules tumorales syngéniques isolée de Tg-MISIIR-Tag-DR26 souris. Objectif: Bien que l&#39;imagerie des tumeurs est possible 7, la détection précoce des tumeurs profondes est difficile chez les animaux vivants de petite taille. Afin de permettre des études précliniques dans un modèle animal immunologiquement intacte pour le cancer ovarien séreux, nous décrivons un modèle de souris syngéniques pour ce type de cancer de l&#39;ovaire qui permet l&#39;imagerie in vivo, les études sur le microenvironnement de la tumeur et une tumeur des réponses immunitaires. Méthodes: Nous avons d&#39;abord tiré une TAG + lignée cellulaire de souris cancéreuses (MOV1) à partir d&#39;une tumeur ovarienne spontanée récoltées dans un 26 semaines, âgé DR26-Tg MISIIR-Tag femelle. Ensuite, nous stablement transduites MOV1 cellules avec TurboFP635 vecteur lentiviral mammifères qui encode Katushka, un mutant rouge lointain de la protéine fluorescente rouge de l&#39;anémone de mer quadricolor Entacmaea avec excitation / émission maxima à 588/635 nm 8,9,10. Nous orthotopique implanté MOV1 Kat dans l&#39;ovaire 11,12,13,14 de non-tumorale sujettes Tg-MISIIR-Tag souris femelles. Progression de la tumeur a été suivie par l&#39;imagerie optique in vivo et microenvironnement de la tumeur a été analysée par immunohistochimie. Résultats: orthotopique implanté MOV1 cellules Kat ont développé des tumeurs de l&#39;ovaire séreux. MOV1 tumeurs Kat pourrait être visualisé par l&#39;imagerie in vivo jusqu&#39;à trois semaines après l&#39;implantation (fig. 1) et ont été infiltrées par les leucocytes, comme observé dans les cancers de l&#39;ovaire humain 15 (fig. 2). Conclusion: Nous décrivons un modèle orthotopique de cancer ovarien adapté à l&#39;imagerie in vivo de tumeurs au début en raison de la haute pH-stabilité et photostabilité de Katushka dans les tissus profonds. Nous proposons l&#39;utilisation de ce nouveau modèle syngénique de cancer ovarien séreux dans les études d&#39;imagerie in vivo et le suivi des réponses immunitaires des tumeurs et d&#39;immunothérapies.

Watch this Scientific Journal Video about An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses at JoVE.com

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Pour étudier<em&gt; In vivo</emLa croissance tumorale&gt; et microenvironnement de la tumeur, nous avons utilisé un modèle de souris syngéniques et orthotopique de cancer ovarien chez les animaux immunocompétents. Nous transduites une lignée de cellules de souris de tumeurs (MOV1) avec Katushka protéine fluorescente (MOV1<sup&gt; KAT</sup&gt;) Et ici nous montrent son implantation orthotopique dans l&#39;ovaire et de<em&gt; In vivo</em&gt; Imagerie.

Un modèle orthotopique de cancer ovarien séreux dans des souris immunocompétentes In vivo L&#39;imagerie des tumeurs et le suivi des tumeurs réponses immunitaires

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

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JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

18.1K vues.  University of Pennsylvania-School of Medicine. Pour étudier In vivo et microenvironnement de la tumeur, nous avons utilisé un modèle de souris syngéniques et orthotopique de cancer ovarien chez les animaux immunocompétents. Nous transduites une lignée de cellules de souris de tumeurs (MOV1) avec Katushka protéine fluorescente (MOV1 KAT) Et ici nous montrent son implantation orthotopique dans l'ovaire et de In vivo Imagerie.

Vidéo: An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modèle de mesure de réponses immunitaires contre le paludisme dans le contexte du VIH co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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Immunologie et infection

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Un modèle de souris transmissible transitoirement IL-8 pour le<em&gt; In vivo</em&gt; Suivi à long terme des réponses inflammatoires

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolement de Salmonella typhimurium-contenant des Phagosomes des Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Adaptation des dosages de cytotoxicité in vivo à l’étude de l’immunodominance dans des réponses cellulaires spécifiques à la tumeur et aux lymphocytes T

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Implantation et suivi par PET/CT d'un modèle orthotopic du mésothéliome pleural humain chez les souris athymiques

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

Visualisation, quantification et cartographie des populations de cellules immunitaires dans le microenvironnement tumoral

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

Cartographie précise du cerveau pour effectuer l’imagerie in vivo répétitive de la dynamique neuro-immune chez les souris

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Modèles de co-culture microfluidique pour disséquer la réponse immunitaire dans des microenvironnements tumoraux in vitro

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infection récurrente des voies urinaires à Escherichia coli déclenchée par Gardnerella vaginalis Exposition à la vessie chez la souris

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.