Reconocemos el apoyo del Instituto Nacional del Cáncer (NCI) R01 CA128908 y Stanford Medical Research Scholar Fellowship. Ningún otro conflicto de intereses pertinentes a este artículo se informó.

1. Modelo de tumor <ol><li> Cultura células H460 (American Type Culture Collection) en medio RPMI 1640 suplementado con suero fetal bovino al 10% y 1% de penicilina / estreptomicina (Invitrogen Life Technologies). Cabe señalar que la elección de líneas de células, medios de cultivo, la ubicación de la inoculación, el número de xenoinjertos por ratón, y otras consideraciones son todos que se adaptan a los objetivos de un estudio particular. Aquí sólo presentamos un diseño de proyecto específico para servir como una ilustración. </li><li> Mantener líneas celulares en una atmósfera humidificada de 5% de CO 2 a 37 ° C y el cambio a un medio fresco cada dos días. </li><li> Cuando una monocapa de 75% confluente de células se forma, separar la monocapa con tripsina y las células se disocian en una sola suspensión de células para el cultivo celular. </li><li> Suspender aproximadamente 1 × 10 6 células H460 en salina tamponada con fosfato (PBS; Invitrogen) y el implante por vía subcutánea enhombros izquierdo y derecho de ratones desnudos (ratones hembra desnudos atímicos (nu / nu), 4 - 6 semanas de edad, Charles River Laboratories, Inc.). </li><li> Permitir el crecimiento de tumores de 150 - 200 mm 3. Se tarda aproximadamente 2 semanas para xenoinjertos de tumor H460 creciendo a este tamaño. Estándar pinza de medición se lleva a cabo para realizar un seguimiento tamaños tumorales. </li><li> Cuando los tumores alcanzan el tamaño ideal de los ratones portadores de tumores son ahora listo para el tratamiento y la visualización in vivo tanto a través de PET y CLI. </li></ol> 2. PET <ol><li> Lleve a cabo los estudios de PET de acuerdo con este programa o cualquier variación de la misma en función del proyecto específico (Figura 1) 12. Un número de factores pueden influir en el diseño de la programación, incluyendo, pero no limitado a, la elección de líneas tumorales de xenoinjertos de células, fármacos contra el cáncer y los regímenes de dosificación. Aquí sólo se presentará un programa de formación de imágenes específico. Los estudios de la CLI se realizó de acuerdo con lahorario mismos que los de los estudios de PET, con CLI realiza inmediatamente después de la PET correspondiente. Cabe también señalar aquí que el propósito de los estudios de PET es principalmente para la validación de los resultados de la CLI. Para los usuarios comunes que sólo desean utilizar instrumentos de OI para sondas de imagen radiomarcados, no es necesario PET. Sin embargo, si se realiza la validación de PET deseo hay que destacar que los instrumentos de PET y CLI debe estar ubicado, muy cerca de la validación de tener éxito debido a la corta vida media de 18 F (109,77 min). </li><li> Divida a los ratones en grupos de tratamiento y control (n ≥ 3 cada uno). El tratamiento de ratones en el grupo de tratamiento con 2 inyecciones de bevacizumab de 20 mg / kg en los días 0 y 2. Día 0 se define por la primera inyección. Tenga en cuenta que en el Día -1 una exploración previa se debe realizar tanto a través de PET y CLI. </li><li> De pequeños animales PET de ratones portadores de tumores se va a realizar con un escáner modelo R4 roedor (Siemens Medical Solutions EE.UU.Un Inc.,). </li><li> Anestesiar todos los ratones con 2% de isoflurano (Aerrane, Baxter) y se inyecta con 3&#39;-desoxi-3&#39;-18 F-fluorotimidina (18 F-FLT; 7,3 a 8,0 MBq [198 a 215 Ci]) a través de la vena de la cola. La sonda de PET es para ser diluido en PBS antes de la inyección. </li><li> Después de 1 h, se anestesian ratones de nuevo y colocar los ratones anestesiados propenso y cerca del centro del campo de visión del escáner PET de pequeños animales. </li><li> Obtener tres minutos exploraciones estáticas y reconstruir las imágenes de un 2-dimensional ordenada subconjuntos algoritmo de máxima expectación. Corrección de fondo no es necesario. </li><li> Dibuje las regiones de interés (ROIs, 5 píxeles para cortes coronales y transaxial) en los tumores en los corregidas caries de cuerpo entero imágenes coronales. Obtener las cuentas máximas por píxel por minuto a partir de las ROIs y convertir a los recuentos por mililitro por minuto mediante el uso de una constante de calibración. Con el supuesto de una densidad de tejido de 1 g / ml, convertir las ROIs a recuentospor gramo por minuto. Determinar la imagen ROI derivados de% ID / g valores dividiendo los recuentos por gramo por minuto por dosis inyectada. Corrección de la atenuación no es necesario. </li></ol> 3. CLI <ol><li> CLI es que se realiza con un sistema de espectro IVIS (Caliper Life Sciences). Adquisición y análisis de imágenes se llevó a cabo utilizando Living Image 3,0 software (Caliper Life Sciences). Longitud de onda, la imagen espectral se realiza utilizando un 18-conjunto de banda estrecha filtro de emisión (490 - 850 nm). Nuevamente, para cada ratón, inmediatamente después de realizar CLI PET para reducir al mínimo la cantidad de desintegración radiactiva si los estudios de PET están incluidos en el protocolo. </li><li> Colocar los animales en una cámara hermética a la luz bajo anestesia isoflurano. Ratones múltiples se pueden colocar simultáneamente para aumentar el rendimiento. </li><li> Adquirir imágenes con tiempo de exposición 3 min (f / stop = 1, hurgar en la basura = 4). Utilice los ajustes de iluminación mismas (tensión de la lámpara, filtros, f / stop, campos de visión, binning) para adquirir todas las imágenes. Utilice el área de piel dorsal para calcular la intensidad de la señal de fondo del tejido. Normalizar la emisión de fluorescencia de fotones por segundo por centímetro cuadrado por estereorradián (p / s / cm 2 / sr). </li></ol> 4. Los resultados representativos La comparación visual entre CLI y las imágenes de PET puede llevarse a cabo fácilmente. Después de la unificación de la barra de escala a través de imágenes de la misma modalidad y lugar de CLI y PET imágenes lado a lado se puede ver en este panel representativo (Figura 2) que tanto CLI y PET revelaron señales de una disminución significativa de H460 xenoinjertos en ratones tratados de pre-tratamiento a 3 días, lo que sugiere un efecto terapéutico significativo. Como comparación, aumenta moderadamente a las señales sin cambios se observaron en los ratones no tratados durante el mismo período de tiempo (datos no mostrados). Por inspección visual solo se puede observar que existe una buena consistencia entre contrastes tumorales que son visualeszado desde CLI y PET. De hecho, esta correlación visual tiene suficiente resolución para mostrar necrosis central del tumor secundario al régimen de tratamiento contra el cáncer (por favor, comparar el CLI y las imágenes de PET a partir del día 3). Para validar los hallazgos de imagen cuantificaciones y análisis de correlación puede llevarse a cabo. Cuantificaciones de CLI y las imágenes PET y un montaje sencillo mediante regresión lineal mostró que las dos modalidades de hecho tenía una excelente correlación (Figura 2 B, R 2 = 0,9309 para 18 F-FLT sondeado grupo de tratamiento). Cabe destacar que, en todos nuestros CLI y estudios de formación de imágenes de PET con diferentes modelos de tumores y diferentes fármacos anticancerígenos las pendientes de los ajustes son también muy cerca, lo que sugiere un excelente ajuste de regresión lineal incluso de todos los datos se conglomerado (datos no mostrados). Ambas imágenes representativas son una adaptación de nuestra publicación anterior 12. er.within-page = &quot;always&quot;&gt; <img src="/files/ftp_upload/4341/4341fig1.jpg" alt="Figura 1"/> Figura 1. Esquema del diseño experimental de los estudios de PET y CLI. Los tumores se implantaron bilateralmente en la región del hombro y se dejó crecer a 150-200 mm 3, y ratones portadores de tumores fueron sometidos a formación de imágenes in vivo a través de PET y CLI en día -1, 1, y 3. Bevacizumab tratamiento se realizó mediante 2 inyecciones de 20 mg / kg en los días 0 y 2. <img src="/files/ftp_upload/4341/4341fig2.jpg" alt="Figura 2"/> Figura 2. (A) En vivo CLI y las imágenes de PET de ratones con H460 xenoinjertos tratados con Bevacizumab antes del tratamiento (pre-scan) y después del tratamiento (día 3). (B) correspondiente análisis cuantitativo de la CLI y PET resultados (n = 3) y sus correlaciones. Imágenes adaptado de (6).arge.jpg &quot;target =&quot; _blank &quot;&gt; Haga clic aquí para ampliar la figura.

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	<li>Weissleder, R., Mahmood, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging.">Molecular imaging.</a> Radiology. 219 (2), 316 (2001).</li><li>Chen, K., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positron+emission+tomography+imaging+of+cancer+biology:+current+status+and+future+prospects.">Positron emission tomography imaging of cancer biology: current status and future prospects.</a> Semin. Oncol. 38 (1), 70 (2011).</li><li>Solomon, M., Liu, Y., Berezin, M. 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S., Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+Cerenkov+light+generation+from+positron-emitting+radiotracers.">Optical imaging of Cerenkov light generation from positron-emitting radiotracers.</a> Phys. Med. Biol. 54 (16), N355 (2009).</li><li>Xu, Y., Liu, H., Cheng, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+radionuclides+for+optical+imaging:+Cerenkov+luminescence+imaging.">Harnessing the power of radionuclides for optical imaging: Cerenkov luminescence imaging.</a> J. Nucl. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+radiation+produced+by+electrons+moving+in+a+medium+with+velocities+exceeding+that+of+light.">Visible radiation produced by electrons moving in a medium with velocities exceeding that of light.</a> Phys Rev. 52 (4), 0378 (1937).</li><li>Boschi, F., Calderan, L., D'Ambrosio, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+18F-FDG+tumour+uptake+measurements+in+small+animals+using+Cerenkov+radiation.">In vivo 18F-FDG tumour uptake measurements in small animals using Cerenkov radiation.</a> Eur. J. Nucl. Med. Mol. 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No hay conflictos de interés declarado.

CLI se está convirtiendo en una técnica de imagenología molecular prometedora que se ha encontrado en muchas potenciales aplicaciones básicas de investigación en ciencias e incluso el uso clínico 4,5,15,16,17. Las principales ventajas de la CLI sobre las tradicionales modalidades de imágenes nucleares como madre de PET de la utilización de instrumentos de OI, que son más fáciles de usar, que se caracteriza por el tiempo de adquisición a corto y alto rendimiento, mucho menos costosas y más accesibl...

<table border="1"&gt;<tr&gt;<td<strong&gt; Nombre</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;</tr&gt;<tr&gt;<td&gt; H460 Línea Celular</td&gt;<td&gt; American Type Culture Collection</td&gt;<td&gt; Número ATCC: HTB-177</td&gt;</tr&gt;<tr&gt;<td&gt; RPMI 1640 Medium</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 12633-012</td&gt;</tr&gt;<tr&gt;<td&gt; Suero Bovino Fetal</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 10091-148</td&gt;</tr&gt;<tr&gt;<td&gt; Penicilina / Estreptomicina</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 15640-055</td&gt;</tr&gt;<tr&gt;<td&gt; Phosphate-Buffered Saline</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 10010-023</td&gt;</tr&gt;<tr&gt;<td&gt; Femenino ratones desnudos atímicos</td&gt;<td&gt; Charles River Laboratories, Inc.</td&gt;<td&gt; Strain Código: 088</td&gt;</tr&gt;<tr&gt;<td&gt; El bevacizumab (Avastin)</td&gt;<td&gt; Genentech / Roche</td&gt;<td&gt; N / A</td&gt;</tr&gt;<tr&gt;<td&gt; MicroPET roedores R4</td&gt;<td&gt; Siemens Medical Solutions EE.UU., Inc.</td&gt;<td&gt; N / A</td&gt;</tr&gt;<tr&gt;<td&gt; Isoflurano (Aerrane)</td&gt;<td&gt; Baxter</td&gt;<td&gt; Baxter Número: AHN3637</td&gt;</tr&gt;<tr&gt;<td&gt; IVIS Spectrum</td&gt;<td&gt; Pinza Ciencias de la Vida</td&gt;<td&gt; N / A</td&gt;</tr&gt;</table

cerenkov luminescence imaging (cli) for cancer therapy monitoring

En la imagen molecular, tomografía por emisión de positrones (PET) y óptico de imágenes (OI) son dos de las modalidades 1-3 más importantes y por lo tanto utiliza más ampliamente. PET se caracteriza por su excelente sensibilidad y capacidad de cuantificación mientras OI se caracteriza por no-radiación, bajo costo relativo, la exploración en tiempo corto, de alto rendimiento, y la amplia disponibilidad a los investigadores básicos. Sin embargo, ambas modalidades tienen sus defectos también. PET sufre de pobre resolución espacial y alto coste, mientras que OI está limitado principalmente a aplicaciones preclínicos debido a su limitada penetración en el tejido junto con las señales de dispersión de prominentes óptica a través del espesor de los tejidos vivos. Recientemente, un puente entre el PET y OI ha surgido con el descubrimiento de Luminiscencia Cerenkov Imaging (CLI) 4-6. CLI es una nueva modalidad de imagen que aprovecha la radiación Cerenkov (CR) para los radionucleidos de imagen con instrumentos de OI. Nobel ruso laureate Alekseyevich Cerenkov y sus colegas descubrieron CR originalmente en 1934. Es una forma de radiación electromagnética emitida cuando una partícula cargada viaja a una velocidad superluminal en un medio dieléctrico 7,8. La partícula cargada, si positrón o electrón, perturba el campo electromagnético del medio mediante el desplazamiento de los electrones en sus átomos. Después de pasar la interrupción de los fotones son emitidos como los electrones desplazados volver al estado fundamental. Por ejemplo, un 18 decaimiento F se calculó para producir un promedio de 3 fotones en agua 5. Desde su aparición, la CLI se ha investigado para su uso en una variedad de aplicaciones incluyendo preclínicos en formación de imágenes de tumores in vivo, del gen de imágenes, radiotrazador desarrollo, multimodalidad de imágenes, entre otros 4,5,9,10,11. La razón más importante por la CLI ha disfrutado de mucho éxito hasta ahora es que esta nueva tecnología se aprovecha de la cooperación bajost y la amplia disponibilidad de OI a los radionucleidos de imagen, que se utilizan para obtener imágenes sólo por técnicas de imagen más caro y menos disponible nucleares tales como el PET. A continuación, se presenta el método para utilizar CLI para supervisar el tratamiento del cáncer de drogas. Nuestro grupo ha investigado recientemente esta nueva aplicación y validado su viabilidad por un estudio de prueba de concepto 12. Hemos demostrado que la CLI y PET mostraron correlaciones excelentes a través de xenoinjertos de tumores diferentes y sondas de imagen. Esto es coherente con el principio general de que CR CLI esencialmente visualiza los radionucleidos mismas PET. Hemos seleccionado Bevacizumab (Avastin; Genentech / Roche) como nuestro agente terapéutico debido a que es un inhibidor de la angiogénesis conocido 13,14. La maduración de esta tecnología en el futuro próximo puede ser concebido para tener un impacto significativo en el desarrollo de fármacos preclínica, la detección, así como la monitorización de la terapia de los pacientes que reciben tratamientos.

Watch this Scientific Journal Video about Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring at JoVE.com

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Uso de Luminiscencia Cerenkov Imaging (CLI) para el control del tratamiento del cáncer preclínico se describe aquí. Este método aprovecha la radiación Cerenkov (CR) e imagen óptico (OI) para visualizar las sondas marcadas radiactivamente y por lo tanto proporciona una alternativa al PET en el seguimiento terapéutico preclínico y la detección de drogas.

Cerenkov Luminiscencia Imaging (CLI) para la Vigilancia del Cáncer Terapia

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

cerenkov-luminescence-imaging-cli-for-cancer-therapy-monitoring

Research

JoVE Journal

Medicine

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

13.6K vistas.  Stanford University. Uso de Luminiscencia Cerenkov Imaging (CLI) para el control del tratamiento del cáncer preclínico se describe aquí. Este método aprovecha la radiación Cerenkov (CR) e imagen óptico (OI) para visualizar las sondas marcadas radiactivamente y por lo tanto proporciona una alternativa al PET en el seguimiento terapéutico preclínico y la detección de drogas.

Video: Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Monitoreo metástasis tumorales y lesiones osteolíticas con bioluminiscencia y Micro Imágenes de TC

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Investigación

Medicina

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelado y de imagen de 3 dimensiones invasión celular colectiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

Protocolos para la Evaluación de radiofrecuencia Interacciones con nanopartículas de oro y Sistemas Biológicos para la terapia del cáncer de hipertermia no invasiva

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Cerenkov Luminiscencia Imaging de interescapular tejido adiposo marrón

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modelo murino de imagen no invasivas para detectar y vigilar cáncer ovárico de Recurrencia

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

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

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

Dinámica de seguimiento del tumor de pulmón para estereotáctica ablativa Radioterapia Corporal

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

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Un Sistema organotípicos de alto rendimiento para la Caracterización de la sensibilidad a los fármacos de las células del mieloma múltiple primaria

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Imagen Fluorescente combinado del infrarrojo cercano y la tomografía computarizada-Micro para visualizar de manera directa tromboembolia cerebral

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualización Cuantitativa del Infiltrado de Leucocitos en un Modelo Murino de Miocarditis Fulminante por Microscopía de Hoja Ligera

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

Micronización mecánica de Lipoaspirates para la terapia regenerativa

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...