Este trabajo fue apoyado por el NIH P01 AI 068.730 (SNC, NS), el NIH subvención CA016520 / TAPITMAT (NS), la financiación privada de la Fundación Claneil (NS), y el de ovario SPORE subvención a la CMCC y la Universidad de Pennsylvania ( P50 CA83638) y el Fox Chase Cancer Center núcleo Grant (P30 CA06927) (DCC). Los autores agradecen la excelente asistencia técnica del Fondo para el núcleo óptico / Bioluminiscencia dirigido por el Dr. EJ Delikatny en la Universidad de Pennsylvania Secreto, Antonio de la célula madre y Core Xenoinjerto dirigido por el Dr. G. Danet-Desnoyers en la Universidad de Pennsylvania cáncer Centro para el SNC de capacitación para la técnica de inyección ortotópica y Dangaj Denada en la Universidad de Pennsylvania / OCRC para ayudar en la cirugía.

1. Cultivo de células <ol><li> Antes de la inyección ortotópico, la cultura MOV1 células Kat, derivada de los tumores DR26, en un matraz T175 hasta que el 90% confluente. Plan para usar de 1 a 5 millones de células por inyección, lo que requiere de 1 o 2 frascos T175. </li><li> En el día de la inyección, la cosecha de las células y determinar el número de células con un hemocitómetro. </li><li> Una vez que la concentración celular se ha determinado, que sedimenten las células por centrifugación durante 5 minutos a 300 x g a temperatura ambiente. </li><li> Después de la vuelta, volver a suspender las células tienen 1 millón en 10 microlitros de PBS estéril con EDTA 0,002 M </li></ol> 2. Antes de la cirugía <ol><li> Antes de la cirugía, llenar una jeringa de insulina 3/10cc con 1 millón de MOV1-Kat células en 10 microlitros de PBS con EDTA. </li><li> La transferencia de un ratón anestesiado con isofluorano a una compresa caliente. Añadir ungüento para los ojos para evitar la deshidratación del ojo. Luego, inmediatamente inserte la cabeza del animal en un sistema de cono conectada a un vaporizador de isoflurano para entregar la anestesia durante la cirugía. </li><li> Después de la desinfección del sitio de la inyección con alcohol, por vía subcutánea inyectar 5 mg / kg de ketoprofeno, un analgésico antes de la cirugía, con una jeringa de insulina 3/10cc. </li><li> Usando las podadoras, afeitarse la parte izquierda de caudal en el dorso de la unión toracolumbar a la base de la cola animal. Aplicar crema de depilación para eliminar por completo el cabello. A continuación, retire el exceso con una toalla de papel húmeda. </li><li> Una vez que el pelo se ha eliminado, esterilizar el área afeitada con povidona yodada y torundas con alcohol. Luego, coloque un paño quirúrgico en la zona de la incisión. </li></ol> 3. Cirugía <ol><li> Justo antes de la cirugía, ajustar los niveles de vaporizador isoflurano al 1,5%. </li><li> Verificar que el animal está completamente anestesiado por pellizcar la almohadilla plantar. </li><li> A continuación, busque el bazo debajo de la piel. Luego, utilizando tijeras quirúrgicas, hacer una incisión dorsolateral 1-2 cm de largo en la parte superior derecha del bazo. </li><li> Disecar el retroperitoneo. La almohadilla alrededor del ovario de ratón se observó. Use pinzas curvas de captar y exponer la almohadilla de grasa que rodea el ovario de ratón. </li><li> Hidratar el órgano con unas gotas de PBS estéril. </li><li> Use pinzas curvas de entender, se retracte de la posición ... ... y luego se asegura el ovario para la inyección </li><li> Mientras que las palancas del ovario con las pinzas, inyectar 10 microlitros de células tumorales MOV1 Kat en el ovario. Un conocimiento firme voluntad evitar la regurgitación de líquidos o fugas. </li><li> Inmediatamente después de la inyección, la liberación de la tensión ejercida por el fórceps. La perforación en el ovario de forma espontánea debe retractarse y cerrar. </li><li> Usando una sutura absorbible de ácido poliglicólico unido a una aguja, cerrar la herida retro-peritoneo. </li><li> Liberar al animal del cono de la nariz. </li><li> Estire la piel y sellar los bordes de heridas dorsolateral con unas gotas de adhesivo tisular. </li><li> Finalmente, por vía oral administrar 100 microlitros de antibióticos a los animales. Luego se coloca de nuevo en su jaula y el monitor para la recuperación. Mantener al animal de los antibióticos en el agua de beber durante una semana. </li></ol> 4. Imágenes in vivo <ol><li> Una semana después de la inyección ortotópico de células tumorales MOV1 Kat, realizar imágenes in vivo. Comience por la transferencia de un ratón anestesiado con isofluorano a la cámara de imágenes. </li><li> Baje el volumen del vaporizador isoflurano al 2%. </li><li> Realizar imágenes in vivo de acuerdo a las instrucciones del fabricante del sistema de imágenes de. En este video, el sistema de Lumina se utilizará. </li><li> A la imagen, haga clic en la imagen viva de software icono en el escritorio. Luego, en el Panel de control IVIS adquisición, seleccione &quot;Ejecutar&quot;. Los ajustes de los instrumentos son análogos a una configuración de la cámara </li><li> En el Panel de Control de la adquisición de configurar los parámetros del instrumento de adquisición. De fluorescencia, marca &quot;fluorescentes&quot;. Haga clic en la casilla de Fotografía para adquirir una fotografía con cada imagen. </li><li> A continuación, establezca el tiempo de exposición como Auto. En &quot;pixel binning o resolución CCD&quot;, marque &quot;Medium&quot;. A continuación, en F / parada o apertura, comprobar el valor de 2. A continuación, seleccione el filtro de excitación de 535 y el filtro de emisiones DsRed. </li><li> En el campo de visión, haga clic en Ver B a la imagen de un ratón. </li><li> A continuación, haga clic en adquirir para iniciar la adquisición de la imagen. </li><li> Una vez que la adquisición de la imagen es completa, el uso de la Región de interés o retorno de la inversión, la herramienta de medida de la señal. Haga clic en el icono de medida para liberar los valores de la señal y el área. </li><li> Por último, haga clic en &quot;Guardar&quot; para guardar la imagen en la carpeta de usuario </li><li> Después de las imágenes se han guardado, interrumpa la administración de isoflurano y devolver el ratón a su jaula. El ratón debe despertar de inmediato. </li></ol> 5. Resultados representante -El uso de este protocolo, El crecimiento in vivo de un cáncer de ovario ortotópico puede ser monitoreado por lo menos 3 semanas con un procedimiento no invasivo. <img src="/files/ftp_upload/2146/2146fig1.jpg" alt="Figura 1" /> Figura 1. MOV1 células Kat, o PBS como control negativo, se inyectaron ortotópicamente en el ovario de no tumorales ratones propensos (derecho de los animales y de los animales a la izquierda y, respectivamente). In vivo de imágenes se realizó después de 2 semanas. La emisión de fluorescencia generada por las células MOV1 Kat injertado en el ovario se midió y comparó con la de ratones control negativo. <img src="/files/ftp_upload/2146/2146fig2.jpg" alt="Figura 2" /> Figura 2. Las secciones congeladas de tumores de ovario MOV1 Kat fueron teñidas con biotina anti-CD4 mAb de seguido de sustrato DAB (marrón oscuro) para detectar la infiltración tumoral de linfocitos (flecha negro). Las células fueron contrastados con metil-verde para visualizar los núcleos de células (azul). Las células tumorales (flechas rojas) fueron morfológicamente distinta de las células T. La diapositiva aparece magnificado 40x.

<ol>
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Cirugía y las inyecciones ortotópico Inyección ortotópica en bolsa ovárica demandas de formación y precisión. Así <ol><li> En el caso de los pobres de la experiencia práctica quirúrgica, con cadáveres en primer lugar. </li><li> Usar preferentemente mujeres multíparas (uno o dos litros) a medida que desarrollan los ovarios más grandes con el tiempo que facilita la inyección y aumentar la supervivencia de comparar con las mujeres nulíparas. </li><li> Debido al pequeño tamaño...

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<td>DMEM-GLUTAMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10564-011</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14040</td>
<td>&nbsp;</td>
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<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>
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<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>06-669-62</td>
<td>&nbsp;</td>
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<td>Artificial tears ointment </td>
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<td>Phoenix Pharma., Inc.</td>
<td>17845-153</td>
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<td>Ketoprofen</td>
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<td>Fort Dodge laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>3cc/insulin syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309301</td>
<td>&nbsp;</td>
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<td>Polyg Polyglycolic Acid suture/needle (3/8 19mm)</td>
<td>&nbsp;</td>
<td>Syneture</td>
<td>9612-31</td>
<td>&nbsp;</td>
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<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>Vetbond</td>
<td>3M</td>
<td>&nbsp;</td>
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<td>Vet Bactrim/ oral suspension</td>
<td>&nbsp;</td>
<td>Hi-tech Pharmacal</td>
<td>840823</td>
<td>&nbsp;</td>
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<td>IVIS-Lumina</td>
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<td>Invitrogen</td>
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<td>Gibco</td>
<td>15140</td>
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<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>
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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

Antecedentes: El cáncer de ovario se diagnostica generalmente en una etapa avanzada, donde la proporción de casos / mortalidad es alta y por lo tanto sigue siendo el más letal de todos los cánceres ginecológicos entre los EE.UU. las mujeres 1,2,3. Los tumores serosos son las formas más generalizadas de cáncer de ovario y el 4,5 transgénicos Tg-MISIIR-TAG representa el modelo de ratón sólo que de forma espontánea se desarrolla este tipo de tumores. Tg-MISIIR-TAG ratones expresan SV40 región transformación bajo control de la sustancia inhibitoria de Müller tipo de receptor II (MISIIR) promotor del gen 6. Otras líneas transgénicas se han identificado que expresan el transgén etiqueta SV40, pero no desarrollan tumores en los ovarios. No tumorales ratones propensos exhibición típica vida de los ratones C57BL / 6 y son fértiles. Estos ratones se puede utilizar como receptores de aloinjertos singénico de células tumorales aisladas de Tg-MISIIR-Tag-DR26 ratones. Objetivo: A pesar de imagen del tumor es posible 7, la detección temprana de los tumores profundos es un reto en animales vivos pequeños. Para permitir que los estudios preclínicos en modelos animales inmunológicamente intactos para el cáncer de ovario seroso, se describe un modelo de ratón singénico para este tipo de cáncer de ovario, que permite imágenes in vivo, los estudios del microambiente del tumor y el tumor de la respuesta inmune. Métodos: En primer lugar, obtuvo un tag + ratón línea celular de cáncer (MOV1) de un tumor ovárico espontáneo recolectadas en un 26 semanas de edad Tg-DR26 MISIIR-Tag mujeres. Luego, de forma estable transduced MOV1 células con el vector Lentivirus TurboFP635 mamíferos que codifica Katushka, un mutante rojo lejano de la proteína fluorescente roja de la anémona de mar Entacmaea quadricolor con excitación / emisión máxima a 588/635 nm 8,9,10. Hemos implantado ortotópicamente MOV1 Kat en el ovario 11,12,13,14 de no tumorales propensos Tg-MISIIR-TAG ratones hembra. La progresión del tumor fue seguido por imágenes in vivo óptica y microambiente tumoral se analizó mediante técnicas de inmunohistoquímica. Resultados: ortotópicamente implantado células MOV1 Kat desarrollaron tumores serosos de ovario. MOV1 tumores Kat puede ser visualizado por imágenes in vivo de hasta tres semanas después de la implantación (fig. 1) y se infiltraron con los leucocitos, como se observa en el cáncer de ovario humano 15 (fig. 2). Conclusiones: Se describe un modelo ortotópico de cáncer de ovario adecuado para imágenes in vivo de tumores antes de tiempo debido a los altos de pH estabilidad y fotoestabilidad de Katushka en los tejidos profundos. Se propone el uso de este nuevo modelo singénico de cáncer de ovario seroso en los estudios de imágenes in vivo y seguimiento de los tumores la respuesta inmune e inmunoterapias.

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

Para el estudio de<em&gt; In vivo</em&gt; El crecimiento del tumor y el microambiente del tumor, se utilizó un modelo de ratón y singénico ortotópico de cáncer de ovario en los animales inmunocompetentes. Nos transducidas un tumor de ratón línea celular (MOV1) con Katushka proteína fluorescente (MOV1<sup&gt; KAT</sup&gt;) Y aquí se muestra su implantación ortotópica en el ovario y<em&gt; In vivo</em&gt; Imágenes.

Un modelo ortotópico de cáncer de ovario seroso en ratones inmunocompetentes para In vivo Imágenes y seguimiento de tumores de las respuestas inmunitarias del tumor

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 ...

an-orthotopic-model-serous-ovarian-cancer-immunocompetent-mice-for

Research

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 vistas.  University of Pennsylvania-School of Medicine. Para el estudio de In vivo El crecimiento del tumor y el microambiente del tumor, se utilizó un modelo de ratón y singénico ortotópico de cáncer de ovario en los animales inmunocompetentes. Nos transducidas un tumor de ratón línea celular (MOV1) con Katushka proteína fluorescente (MOV1 KAT) Y aquí se muestra su implantación ortotópica en el ovario y In vivo Imágenes.

Video: 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; Modelo para medir la respuesta inmune a la malaria en el contexto del VIH Co-infección

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

Investigación

Inmunología e Infección

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 modelo de ratón IL-8 transgénicamente transgéni-<em&gt; In Vivo</em&gt; Monitoreo a Largo Plazo de las Respuestas Inflamatorias

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.

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

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.

Confección de ensayos de citotoxicidad in vivo para estudiar inmunodominancia en respuestas de células T CD8+ específicas de tumores

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.

Implantación y monitorización por PET/CT de un modelo ortotópico de mesotelioma pleural humano en ratones atímicos

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.

Visualización, cuantificación y mapeo de poblaciones de células inmunes en el microambiente 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.

Mapeo cerebral preciso para realizar imágenes in vivo repetitivas de dinámica neuroinmunitaria en ratones

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.

Modelos de cocultivo microfluídico para diseccionar la respuesta inmune en microambientes tumorales 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.

Infección recurrente del tracto urinario por Escherichia coli desencadenada por la exposición de la vejiga de Gardnerella vaginalis en ratones

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.