Reconhecemos o apoio do Instituto Nacional do Câncer (NCI) R01 CA128908 e Stanford Medical Research Scholar Fellowship. Nenhum outro conflito potencial de interesse relevante para este artigo foi relatado.

1. Modelo de Tumor <ol><li> Cultura de células H460 (American Type Culture Collection) em meio RPMI 1640 suplementado com 10% de soro fetal bovino e 1% de penicilina / estreptomicina (Invitrogen Life Technologies). Deve notar-se que as escolhas de linhas celulares, meios de cultura, os locais de inoculação, o número de xenoenxertos por rato, e outras considerações são todos de ser adaptado para os objectivos de um determinado estudo. Aqui vamos apresentar apenas um projeto de projeto específico para servir como uma ilustração. </li><li> Manter as linhas de células numa atmosfera humidificada de 5% de CO2 a 37 ° C e de mudança para meio fresco todos os dias. </li><li> Quando uma monocamada confluente a 75% das células é formada, separar a monocamada com tripsina e dissociam-se as células numa suspensão de células individuais para a cultura da célula. </li><li> Suspender a cerca de 1 x 10 6 células H460 em tampão fosfato salino (PBS; Invitrogen) e o implante por via subcutânea emombros esquerdo e direito de ratinhos nus do sexo feminino (ratinhos nus atímicos (nu / nu), 4 - 6 semanas de idade, Charles River Laboratories, Inc.). </li><li> Permitir o crescimento dos tumores de 150-200 mm 3. Demora cerca de 2 semanas para xenoenxertos de tumores H460 a crescer a este tamanho. Padrão paquímetro medição é realizada para acompanhar os tamanhos tumorais. </li><li> Quando os tumores atingem o tamanho ideal os ratos portadores do tumor está agora pronto para o tratamento in vivo e imagiologia por PET e CLI. </li></ol> 2. PET <ol><li> Realizar os estudos de PET de acordo com esse cronograma ou qualquer variação do mesmo, dependendo do projeto específico (Figura 1) 12. Um certo número de factores podem influenciar o desenho da agenda, incluindo, mas não limitado a, a escolha de linhas de xenoenxertos de células de tumor, fármacos anti-cancerígenos, e regimes de dosagem. Aqui vamos apenas apresentar um cronograma de imagem específico. Os estudos da CLI devem ser realizadas de acordo com omesmo esquema como os dos estudos de PET, com CLI realizada imediatamente após o PET correspondente. Deve ser também notado aqui que o objectivo dos estudos de PET-se principalmente para a validação dos resultados de CLI. Para usuários comuns, que só querem usar instrumentos OI para testes de imagem radiomarcados, não PET é necessário. No entanto, se faz uma validação PET desejo deve sublinhar-se que os instrumentos de PET e CLI deve ser localizada dentro de uma proximidade muito estreita para a validação para ser bem sucedido por causa da meia-vida curta de 18 F (109,77 min). </li><li> Divida ratos em grupos de tratamento e controle (n ≥ 3 cada). Tratar os ratos no grupo de tratamento com 2 injecções de bevacizumab de 20 mg / kg nos dias 0 e 2. Dia 0 é definido pela primeira injecção. Note-se que no dia -1 um pré-scan deve ser realizada através de PET e CLI. </li><li> Pequenos animais PET de ratos portadores de tumores é para ser realizado com um aparelho de modelo de roedor R4 (Siemens Medical Solutions EUAA Inc.,). </li><li> Anestesiar os ratos com 2% de isoflurano (Aerrane, Baxter) e injectar com 3&#39;-desoxi-3&#39;-18 F-fluorotimidina (18 F-FLT; 7,3-8,0 MBq [198-215 | iCi]) através da veia da cauda. A sonda de PET deve ser diluído em PBS antes da injecção. </li><li> Depois de 1 hora, anestesiar ratos e coloque novamente anestesiados ratos propensos e próximo do centro do campo de visão do scanner PET pequenos animais. </li><li> Obter três minutos exames estáticos e reconstruir as imagens por um 2-dimensional algoritmo de máxima ordenada subconjuntos expectativa. Correção de fundo não é necessário. </li><li> Desenhe regiões de interesse (ROIs; 5 pixels para cortes coronais e transaxial) nos tumores no decaimento-corrigidas de corpo inteiro imagens coronais. Obter as contagens máximas por pixel por minuto a partir da ROIs e converter em unidades de contagem por mililitro por minuto por utilização de uma constante de calibração. Com o pressuposto de uma densidade de tecido de 1 g / ml, para converter o ROIs contagenspor grama por minuto. Determinar ROI imagem derivados% ID / g valores dividindo contagens por grama por minuto pelo dose injectada. Correção de atenuação não é necessário. </li></ol> 3. CLI <ol><li> CLI é para ser realizado com um sistema IVIS Spectrum (Caliper Life Sciences). A aquisição e análise de imagens são para ser efectuados utilizando Vivo Imagem 3.0 software (Caliper Life Sciences). Wavelength-resolved imagem espectral é para ser realizado utilizando um conjunto de 18 de emissão de banda estreita do filtro (490-850 nm). Mais uma vez, para cada rato, imediatamente depois de executar CLI PET para minimizar a quantidade de decaimento radioactivo se os estudos de PET são incluídos no protocolo. </li><li> Coloque os animais em uma câmara à prova de luz com anestesia isoflurano. Ratinhos múltiplos podem ser colocados simultaneamente para aumentar o rendimento. </li><li> Aquisição de imagens utilizando o tempo de exposição 3 min (f / stop = 1 binning, = 4). Use as configurações de iluminação mesmo (tensão da lâmpada, filtros, f / stop, campos de visão, binning) para adquirir todas as imagens. Usar a área da pele dorsal para calcular a intensidade do sinal do tecido de fundo. Normalizar emissão de fluorescência de fótons por segundo por centímetro quadrado por esterradiano (p / s / cm 2 / sr). </li></ol> 4. Resultados representativos Comparação visual entre CLI e imagens de PET podem ser facilmente realizados. Após a barra de unificar escala através de imagens da mesma modalidade e local CLI e PET imagens lado a lado, uma pode ver neste painel representativo (Figura 2A) que tanto CLI e PET revelou sinais diminuiu significativamente de H460 xenoenxertos em camundongos tratados de pré-tratamento ao dia 3, o que sugere efeito terapêutico significativo. Como comparação, moderadamente aumentado de sinais inalterados foram observados em ratinhos não tratados, durante o mesmo período de tempo (dados não mostrados). Por inspecção visual sozinho pode-se observar que existe uma boa consistência entre contrastes tumorais que são visualzada de CLI e PET. Na verdade, essa correlação visual tem resolução suficiente para mostrar necrose central do tumor secundário para o regime de tratamento anticâncer (por favor, comparar o CLI e imagens de PET a partir do dia 3). Para validar os resultados das quantificações de imagem e análise de correlação pode ser realizada. Quantificações de CLI e imagens de PET e uma montagem simples através de regressão linear mostrou que as duas modalidades de facto tinha uma excelente correlação (Figura 2B, R 2 = 0,9309 durante 18 F-FLT sondado grupo de tratamento). Notavelmente, em todos os nossos CLI e estudos de imagem PET com diferentes modelos de tumores e de diferentes fármacos anticancerígenos as inclinações das convulsões são também extremamente estreita, sugerindo assim uma excelente combinação de regressão linear mesmo de todos os dados são conglomerados (dados não mostrados). Ambas as imagens representativas são adaptados a partir de nossa publicação anterior 12. er.within-page = &quot;always&quot;&gt; <img src="/files/ftp_upload/4341/4341fig1.jpg" alt="Figura 1"/> Figura 1. Esquemática do delineamento experimental de estudos de PET e CLI. Os tumores foram implantados bilateralmente na região do ombro e deixadas a crescer até 150-200 mm 3, e ratos portadores de tumor foram submetidos a imagiologia in vivo por meio de PET e CLI no dia -1, 1 e 3. Bevacizumab tratamento foi realizado por duas injecções de 20 mg / kg nos dias 0 e 2. <img src="/files/ftp_upload/4341/4341fig2.jpg" alt="Figura 2"/> Figura 2. (A) In vivo CLI e imagens PET de ratinhos portadores de xenoenxertos H460 tratadas com Bevacizumab antes do tratamento (pré-scan) e após o tratamento (dia 3). (B) análise quantitativa correspondente da CLI e PET resultados (n = 3) e os seus correlações. Imagens adaptada a partir de (6).arge.jpg &quot;target =&quot; _blank &quot;&gt; Clique aqui para ver maior figura.

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CLI está emergindo como uma técnica de imagem molecular promissor que tem encontrado em muitos potenciais aplicações das ciências básicas de pesquisa e até mesmo o uso clínico 4,5,15,16,17. As principais vantagens do CLI mais tradicionais modalidades de imagens nucleares, tais como tronco a partir de PET da utilização de instrumentos OI, que são mais fáceis de usar, caracterizados por tempo de aquisição curto e alta taxa de transferência, significativamente menos caras, e mais amplamente dispon...

<table border="1"&gt;<tr&gt;<td<strongNome&gt;</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; Linha Celular H460</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</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 12633-012</td&gt;</tr&gt;<tr&gt;<td&gt; Soro Fetal Bovino</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; Salina tamponada com fosfato</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 10010-023</td&gt;</tr&gt;<tr&gt;<td&gt; Fêmea atímicos camundongos nude</td&gt;<td&gt; Charles River Laboratories, Inc.</td&gt;<td&gt; Código de tensão: 088</td&gt;</tr&gt;<tr&gt;<td&gt; Bevacizumab (Avastin)</td&gt;<td&gt; Genentech / Roche</td&gt;<td&gt; N / A</td&gt;</tr&gt;<tr&gt;<td&gt; Roedor MicroPET R4</td&gt;<td&gt; Siemens Medical Solutions EUA, 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; Número Baxter: AHN3637</td&gt;</tr&gt;<tr&gt;<td&gt; IVIS Spectrum</td&gt;<td&gt; Caliper Life Sciences</td&gt;<td&gt; N / A</td&gt;</tr&gt;</table

cerenkov luminescence imaging (cli) for cancer therapy monitoring

Em imagiologia molecular, positron emission tomography (PET) e imagiologia óptica (OI) são duas das modalidades mais importantes e, portanto, mais amplamente utilizado 1-3. PET é caracterizado pela sua excelente sensibilidade e capacidade de quantificação, enquanto OI é notável para a radiação de não-, relativo baixo custo, o tempo de verificação de curto, com rendimento elevado, e a disponibilidade de largura de pesquisadores básicos. No entanto, ambas as modalidades têm suas deficiências também. PET sofre de resolução espacial pobre e de custo elevado, enquanto que a OI é principalmente limitada a aplicações pré-clínicos devido à sua penetração nos tecidos limitada, juntamente com os sinais de espalhamento proeminentes ópticas através da espessura de tecidos vivos. Recentemente, uma ponte entre o PET e OI surgiu com a descoberta de luminescência de Cerenkov Imaging (CLI) 4-6. CLI é uma nova modalidade de imagem que utiliza radiação de Cerenkov (CR) para radionuclídeos de imagem com instrumentos OI. Nobel russo laurEATE Alekseyevich Cerenkov e seus colegas originalmente descoberto CR em 1934. É uma forma de radiação electromagnética emitida quando uma partícula carregada viaja a uma velocidade superluminal num meio dieléctrico 7,8. A partícula carregada, se pósitron ou elétron, perturba o campo eletromagnético do meio, deslocando os elétrons em seus átomos. Depois de passar dos fótons de ruptura são emitidos como os elétrons deslocados retornar ao estado fundamental. Por exemplo, um decaimento F 18 foi estimada para produzir uma média de 3 fotões em água 5. Desde o seu aparecimento, CLI tem sido investigada para o seu uso numa grande variedade de aplicações, incluindo pré-clínicos em imagiologia de tumores in vivo, do gene repórter de imagem, radiofármaco desenvolvimento, multimodalidade de imagem, entre outros 4,5,9,10,11. A razão mais importante pela qual CLI tem desfrutado de muito sucesso até agora é que esta nova tecnologia aproveita o co baixoº e ampla disponibilidade de OI para radionuclídeos de imagem, que costumavam ser fotografada apenas pelo mais caro e menos disponível modalidades de imagens nucleares, tais como PET. Aqui, apresentamos o método de usar CLI para monitorar câncer de terapia medicamentosa. Nosso grupo foi recentemente investigado esta nova aplicação e validada a sua viabilidade através de um estudo de prova de conceito-12. Nós demonstramos que a CLI e PET apresentaram excelentes correlações através de xenotransplantes tumorais diferentes e sondas de imagem. Isto é consistente com o princípio fundamental da CR que CLI essencialmente visualiza os radionuclídeos mesmas PET. Foram selecionados Bevacizumab (Avastin, Genentech / Roche) como o agente terapêutico porque é um inibidor da angiogénese bem conhecido 13,14. Maturação desta tecnologia no futuro próximo pode ser vislumbrada a ter um impacto significativo no desenvolvimento de medicamentos pré-clínico, triagem, bem como a monitorização terapêutica de pacientes que recebem tratamentos.

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 luminescência de Cerenkov Imaging (CLI) para monitorar o tratamento de câncer pré-clínico é descrito aqui. Este método tira vantagem da radiação Cerenkov (CR) e imagiologia óptica (OI) para visualizar sondas marcadas radioactivamente e, assim, fornece uma alternativa ao PET na monitorização terapêutica pré-clínica e triagem de drogas.

Luminescência de Cerenkov Imaging (CLI) para o Monitoramento Terapia de Câncer

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 visualizações.  Stanford University. Uso de luminescência de Cerenkov Imaging (CLI) para monitorar o tratamento de câncer pré-clínico é descrito aqui. Este método tira vantagem da radiação Cerenkov (CR) e imagiologia óptica (OI) para visualizar sondas marcadas radioactivamente e, assim, fornece uma alternativa ao PET na monitorização terapêutica pré-clínica e triagem de drogas.

Vídeo: 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.

Monitoramento metástases tumorais e lesões osteolíticas com bioluminescência e CT Micro Imagem

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

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.

Modelagem e imagem 3-Dimensional Invasion cela coletiva

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 Avaliação de radiofreqüência Interações com Nanopartículas de Ouro e Sistemas biológicos para terapia não-invasiva Câncer Hipertermia

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 luminescência imagem de Interscapular Brown tecido adiposo

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 imagem não invasivo para detectar e monitorar o cancro do ovário Recorrência

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.

Rastreamento Dinâmico Tumor do pulmão por estereotáxica ablativo Radioterapia do corpo

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.

Um Sistema Organotípicas alta taxa de transferência para Caracterização de Drogas de sensibilidade de células de mieloma múltiplo primárias

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.

Combinada Near-infrared imagens fluorescentes e tomografia Micro-computado para Diretamente Visualizando Cerebral tromboêmbolos

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.

Visualização Quantitativa de Infiltrado de Leucócitos em um Modelo Murino de Miocardite Fulminante por Microscopia de Folha de Luz

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.

Micronização mecânica de Lipoaspirates para terapia regenerativa

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