Este trabalho é apoiado em parte pela American Heart Association GRA Spring 09 Postdoctoral Clube (09POST2250268) eo NIH R01 CA097465. Nós apreciamos muito Dr. Wen Li e Dr. Vikas Gulani para teste protocolo MRI e configuração, ea Sra. Yvonne Parker por sua assistência na implantação do tumor.

Protocolo adaptado de um estudo prévio 11. 1. Conjugação de Gd-DOTA para CLT1 Peptide <ol><li> Usando a síntese de péptidos em fase sólida padrão, sintetizar CLT1 péptido (CGLIIQKNEC) a partir de aminoácidos protegidos com Fmoc numa resina de cloreto de 2-clorotritilo (1,0 mmol). </li><li> Após a adição de aminoácido final, ciclizar o péptido linear em-resina com tálio (III), trifluoroacetato (1,09 g, 2,0 mmol, 2 equivalentes) em DMF (20 ml) a 0 ° C durante 2 horas. </li><li> Em seguida, o conjugado de PEG e lisina sequencialmente para o N-terminal do péptido CLT1 (1,0 mmol) de resina por meio de reacção sequencial com Fmoc-NH-PEG-COOH (3,0 mmol), Fmoc-Lys (Fmoc)-OH (3,0 mmol), e uma segunda porção de Fmoc-Lys (Fmoc)-OH (6 mmol). </li><li> Retirar Fmoc com piperidina. Adicionar DOTA-tris (t-Bu) (4,58 g, 8,0 mmol, 2 equivalentes para cada grupo amino) para reagir com as aminas livres de quatro dendrímero lisina sobre a resina durante 2 horas. </li><li> Finalmente, clivar e desproteger o produtoa partir da resina por tratamento com um cocktail de ácido trifluoroacético, água e triisobutylsilane (TIS) (20 ml, 95/2.5/2.5) durante 8 horas à temperatura ambiente. Remover a resina por filtração, seguida de lavagem com ácido trifluoroacético (TFA). Adicionar os filtrados combinados gota a gota a éter etílico frio (200 ml), centrífuga, e lava-se com éter etílico 4x. Seca-se sob vácuo para dar um sólido incolor (1,99 g, 61% de rendimento). </li><li> Purifica-se o produto em bruto por HPLC preparativa utilizando um gradiente de 0-40% de solvente B (0,1% TFA em acetonitrilo) no solvente A (0,1% de solução aquosa de TFA) durante 20 minutos e 40-90% de solvente B em solvente A durante 10 min . </li><li> Dissolver CLT1-dl-(DOTA) 4 (250 mg, 0,077 mmol) em água Dl (15 ml) e ajustar o pH a 6 usando NaOH 1M. Adicionar Gd (OAc) 3 0,4 H 2 O (472 mg, 0,462 mmol, 1,5 equivalentes a monoamida DOTA), em porções, à solução, enquanto se mantinha o pH a 6 usando NaOH 1M. Agita-se a solução de reacção à temperatura ambiente durante 48 horas. </li><li> Complexo do Gd residual (III) com ethylenediaminetetraacetic ácido acético (EDTA) (90 mg, 0,31 mmol), e purifica-se o produto bruto com uma coluna de exclusão de tamanho para obter o produto final CLT1-dl-(Gd-DOTA) 4 (169 mg, 57%). </li></ol> 2. Caracterização de agentes de contraste <ol><li> Medir o teor de Gd (III), com a espectroscopia de emissão de plasma indutivamente acoplado-óptico. </li><li> Adquirir laser de dessorção / ionização de tempo-de-voo (MALDI-TOF) os espectros de massa assistida por matriz no modo linear com o ácido 2,5-di-hidroxibenzóico (2,5-DHB) como uma matriz. </li><li> Medida do tempo de relaxação da solução aquosa dos agentes de contraste de CLT1-dl-(Gd-DOTA) e 4 um agente de controlo de nontargeted sCLT1-dl-(Gd-DOTA) 4 com diferentes concentrações, a 60 MHz (1,5 T) com um relaxometer a 37 ° C. Use uma seqüência de pulso inversão-recuperação para medir T 1 e uma seqüência de Carr-Purcell-Meiboom-Gill com 500 ecos para medir T 2. Calcule T 1 e T 2 relaxividades of thagentes e das encostas das parcelas de 1 / T 1 e 1 / T 2 versus as concentrações de D&#39;us. </li></ol> 3. Cultura Celular e Desenvolvimento de Modelo Animal com Ortotópico PC3 próstata Tumor <ol><li> Cultura PC3 células de cancro da próstata com a expressão constitutiva da proteína de fluorescência verde (GFP) em meio RPMI suplementado com 5% de soro bovino fetal e penicilina / estreptomicina / fungizona, colheita por tripsinização e ressuspender a densidade de 2,5 x 10 4 células / ul de PBS. </li><li> Manter NIH ratos pelados atímicos machos (4-5 semanas de idade) no Centro de atímicos animal Core, Case Western Reserve University (CWRU) de acordo com um protocolo de animais aprovado pelo Comitê Animal Care e Use Institucional CWRU. </li><li> As superfícies cirúrgicos foram pulverizadas com uma solução de clorexidina a 2% e, em seguida, coberto com cortinas absorventes. Luvas cirúrgicas estéreis foram usados ​​durante o procedimento. Antes da cirurgia animal, ip injetar Avertin (250 mg / kg) para anesthetizenviar um e ratos. Alternativamente, a cetamina / xilazina / acepromazina pode ser utilizado numa concentração de 50/5/1 mg / kg. Pomada oftálmica foi aplicada ao rato para manter a humidade adequada durante a anestesia. </li><li> Comece a cirurgia por corte de uma pequena incisão através da pele e do peritoneu ao longo da linha média inferior a um comprimento de cerca de 1 cm, usando um bisturi estéril. A cortina cirúrgica estéril foi coberto ao redor do local da incisão durante a cirurgia de sobrevivência. </li><li> Suavemente exteriorizar e estabilizar os lóbulos da próstata dorsal para expor a próstata. Injectar a suspensão de células PC3-GFP em PBS (20 mL) na próstata com uma agulha de calibre 30. Finalmente fechar a incisão com ferida Autoclip 12. </li><li> Para o monitoramento pós-procedimento, monitorar os animais duas vezes por dia durante uma semana. Sinais de doença incluem a falta de alimentação, urina escura coleta em torno da uretra, e marcha instável que pode indicar infecção ou obstrução da bexiga nos grampos. </li><li> Após 10 dias, remover o tele grampos usando um removedor de grampos. Monitorizar os animais diariamente para avaliar o tamanho do tumor e qualquer evidência de dor, tais como a perda de peso, os desvios de comportamento, e defeitos de motor. Eutanásia dos animais que apresentaram um tamanho tumoral superior ao limite permitido eticamente ou evidência de dor durante o crescimento do tumor. </li></ol> 4. Confirmação de Tumor Encadernação Especificidade dos Peptídeos com imagens de fluorescência e Histologia <ol><li> Após a inoculação de células tumorais, permitir que cerca de 4 semanas de crescimento do tumor, antes de iniciar a geração de imagens. Antes da injeção das sondas de peptídeos, adquirir imagens de fluorescência de GFP com camundongos vivos em um gerador de imagens de fluorescência para verificar a presença de tumor. </li><li> Iv injectar Texas Red péptidos marcados para a ratinhos portadores do tumor a uma dose de 10 nmol / rato. </li><li> Sacrificar os ratos 2 horas mais tarde por deslocamento cervical, recolher o tumor e órgãos principais, e imediatamente imagem em um gerador de imagens de fluorescência. Use filtros de luz verde para GFP (excitação: 444-490 nm; emissão: 515 nm filtro passa muito tempo; configurações de aquisição: 500-720 em 10 passos nm) e filtros de luz vermelha para o Texas Red (excitação: 576-621 nm; emissão: 635 nm filtro passa muito tempo; aquisição configurações: 630-800 em 10 passos Nm). Tempo de exposição de 10 ms para GFP e 150 ms para vermelho do Texas. </li><li> Imediatamente após a medição da fluorescência do tecido todo, coletar tecidos tumorais, fixar com formalina e criocorte em fatias 5 mícrons. </li><li> Após lavagem com PBS, monte com uma gota de meio de montagem contendo 4 &#39;,6-diamidino-2-phenylindole (DAPI), imagem imediatamente em um microscópio de varredura a laser confocal. </li></ol> 5. Imagem por Ressonância Magnética (MRI) <ol><li> Cerca de 4 semanas após a inoculação de células tumorais, permitem que os tumores cresçam até 0,3-0,6 cm de diâmetro. Use um scanner de ressonância magnética 7 T com um rádio de freqüência de volume (RF) bobina para estudo de ressonância magnética. </li><li> Anestesiar o rato com uma mistura de isoflurano em oxigênio 2% em uma câmara de indução isoflurano. Ophpomada thalmic foi aplicada ao rato para manter a humidade adequada durante a anestesia. </li><li> Inserir uma agulha de calibre 30 em um tubo de 1,0 m de comprimento cheia com solução salina heparinizada. Colocar o cateter de auto-feito na veia da cauda do rato. </li><li> Posicione o mouse para o ímã e manter sob anestesia inalatória com isoflurano a 1,5% em oxigênio através de um cone do nariz. </li><li> Coloque um sensor respiratório conectado a um sistema de monitoramento no abdômen para monitorar a freqüência ea profundidade da respiração. Manter a temperatura do corpo a 37 ° C por um sopro de ar quente para o íman através de um sistema de controlo de realimentação. </li><li> Comece com imagens de cortes sagital usando uma seqüência de localizações para identificar a localização do tumor (TR / TE = 200/3.7 ms, FOV = 3,0 cm, espessura de corte = 2,2 mm, número fatia = 16, média = 2, o ângulo de inclinação = 45 °, matriz = 128 x 128). </li><li> Use um gradiente seqüência supressão de gordura 2D T1-weighted para adquirir imagens axiais em 2D para CE-MRI (TR / TE = 151.2/1.9 ms, FOV = 3,0 centímetros x 3,0 cm, spiolhos espessura = 1,2 mm número fatia = 12, média = 1, o ângulo de inclinação = 80 °, matriz = 128 x 128). </li><li> Depois de pré-injecção de aquisição de imagens MR de linha de base, começam a injectar o agente alvo ou agente de controlo a uma dose de 0,03 mmol de Gd / kg, através de lavagem com 200 mL de solução salina. </li><li> Continuar a aquisição de imagens de ressonância magnética-CE em diferentes pontos de tempo de até 30 min. </li></ol> 6. Processamento de Imagem e Análise <ol><li> Use o software de imagens para análise de imagens. </li><li> Desenhe as regiões de interesse (ROI) em todo o tumor e os rins no plano de imagem bidimensional e medir a intensidade média do sinal. </li><li> Para quantificar os dados CE-MRI, medir tumor ou realce de contraste de rim (ΔSNR) por cálculo do aumento da pós-contraste SNR sobre pré-contraste SNR usando a seguinte equação: ΔSNR = (S t / σ t) - (S 0 / σ 0), em que R 0 e S t denotar o sinal no tumou ou os rins, antes e depois de contraste, e σ 0 e σ t são o desvio padrão do ruído de medida a partir do fundo do ar antes e depois de contraste. </li><li> Calcule os valores de p usando bicaudal do teste t de Student, assumindo significância estatística de p &lt;0,05. </li></ol>

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Etapas críticas Seleção de Biomarcador adequada e Segmentação pequeno peptídeo Para desenvolver com sucesso um agente de contraste com o tamanho pequeno alvo, dois pontos principais devem ser consideradas. Em primeiro lugar, é importante seleccionar os biomarcadores moleculares adequados, que são abundantemente presentes em tecidos doentes com pouca ou nenhuma presença nos tecidos normais. Nossa biomarcador relacionada ao câncer selecionad...

Imagem eficaz de alvos moleculares relacionadas ao câncer é de grande importância para melhorar a precisão de antes detecção e diagnóstico do câncer. A ressonância magnética (RM) é uma modalidade de imagem clínica poderoso com alta resolução espacial e sem radiação de ionização 1. No entanto, nenhum agente de contraste alvo está disponível para imagem molecular do câncer MR clínica. Design inovador e desenvolvimento de agentes de contraste alvo de ressonância magnética avançaria muito a aplicação de imagem molecular do câncer MR. Foram feitos esforços significativos para desenvolver agentes de contraste para ressonância magnética direcionados dos biomarcadores expressas na superfície das células cancerosas. Devido à sensibilidade relativamente baixa ressonância magnética e baixa concentração desses biomarcadores, é um desafio para gerar aumento de contraste suficiente para imagiologia molecular MR eficaz usando pequenos agentes de contraste alvo molecular 2,3. A fim de obter reforço suficiente, vários sistemas de entrega de such como lipossomas, nanopartículas e polímeros conjugados com uma elevada carga de paramagnéticos Gd quelatos (III) foram preparados para aumentar a concentração local de agentes de contraste para os sites de destino 4,5. Embora estes sistemas de entrega eram capazes de gerar melhoria significativa do tumor em modelos animais, os tamanhos grandes resultaram na eliminação lenta e incompleta do corpo, resultando na acumulação prolongada de Gd iões tóxicos (III), que podem causar problemas graves de segurança 6. Recentemente, alguns estudos demonstraram que as limitações de ressonância magnética para imagiologia molecular pode ser superado pela selecção biomarcadores adequados com elevada expressão local das lesões e usando pequenos agentes moleculares que podem ser facilmente excretados 7,8. A principal característica desses agentes é que eles têm como alvo marcadores moleculares abundantemente presentes nos tecidos doentes com pouca presença nos tecidos normais. Uma alta concentração de agentes de contraste pode ligar-se a estes alvos, resultando em sufirealce de contraste ciente para imagiologia molecular MR eficaz. Uma vez que o seu tamanho é mais pequeno do que o limiar de filtração renal, os agentes de contraste não ligados podem ser facilmente eliminados do corpo com a redução do ruído de fundo. Nós selecionamos um biomarcador relacionada ao câncer universal, proteínas do plasma coagulado, que existem em abundância estroma tumoral, e raramente estão presentes em tecidos normais 9. Sintetizamos um agente de contraste de alvo contendo um pequeno péptido de alvejamento CGLIIQKNEC (CLT1), que mostrou uma forte ligação específica para o modelo de tumor de próstata PC3 10, e quatro monoamida quelatos Gd-DOTA. Aqui, nós fornecemos uma metodologia de imagem molecular para detectar câncer MR tumores em camundongos.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Fmoc protected amino acids</td>
			<td>EMD Chemicals Inc</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DOTA-tris(t-Bu)</td>
			<td>TCI America</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PyBOP, HOBt, HBTU</td>
			<td>Nova Biochem</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DIPEA, Thallium(III) trifluoroacetate, TIS</td>
			<td>Sigma-Aldrich Corp.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Texas Red, succinimidyl ester, single isomer</td>
			<td>Invitrogen</td>
			<td>T20175</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENTS</td>
		 </tr>
		 <tr>
			<td>Agilent 1100 HPLC system</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ZORBAX 300SB-C18 PrepHT column</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ICP-OES Optima 3100XL</td>
			<td>Perkin-Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>AutoflexTM Speed</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Maestro FLEX In Vivo Imaging System</td>
			<td>Cambridge Research &amp; Instrumentation, Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biospec 7T MRI scanner</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

mr molecular imaging of prostate cancer with a small molecular clt1 peptide targeted contrast agent

Matriz extracelular do tumor tem abundância de proteínas relacionadas com o cancro que podem ser utilizados como biomarcadores para imagiologia molecular do cancro. Neste trabalho, nós demonstramos imagem molecular do câncer MR eficaz com um pequeno peptídeo molecular complexa monoamida Gd-DOTA apontado como um agente de contraste MRI alvo específico para proteínas do plasma coagulado no estroma tumoral. Realizamos a experiência de avaliar a eficácia do agente para detecção não invasiva de tumor de próstata com ressonância magnética em um modelo ortotópico de câncer de próstata do rato PC-3. O agente de contraste de alvo era eficaz para produzir aumento significativo de contraste do tumor com uma dose baixa de 0,03 mmol de Gd / kg. O peptídeo alvo agente de contraste MRI é promissor para ressonância magnética molecular do tumor da próstata.

A Figura 1 descreve a síntese do agente de contraste alvo CLT1-dl-(Gd-DOTA) 4 e o esquema geral do experimento. CLT1-dl-(Gd-DOTA) 4 mostra muito maior relaxividade do que clínico Gd-DOTA (Tabela 1). A 1,5 T, T 1 relaxividade por gadolínio de CLT1-dl-(Gd-DOTA) 4 em PBS (pH 7,4) é de aproximadamente 3 vezes mais elevada do que a de Gd-DOTA 10. Imagiologia Maestro confirma a forte ligação específica de vermelho do Texas marcado CLT1 (CLT1-TR) de...

Watch this Scientific Journal Video about MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent at JoVE.com

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Para demonstrar imagem molecular do câncer MR com um pequeno peptídeo alvo MRI agente de contraste específico para proteínas do plasma coagulado no estroma tumoral em um modelo de câncer de próstata do rato.

MR Imagem Molecular do cancro da próstata com um pequeno Molecular CLT1 Peptide alvejado agente de contraste

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

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11.1K visualizações.  Case Western Reserve University.  Para demonstrar imagem molecular do câncer MR com um pequeno peptídeo alvo MRI agente de contraste específico para proteínas do plasma coagulado no estroma tumoral em um modelo de câncer de próstata do rato. 

Vídeo: MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

In vivo Near Infrared fluorescência de imagem intravascular (NIRF) Molecular da placa inflamatória, uma Abordagem Multimodal para imagem da Aterosclerose

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Medicina

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Contraste de imagem aprimorada navio usando MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Avaliação de Captação de nanopartículas em tumores em tempo real usando imagens intravital

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Imagem Molecular para atingir as células progenitoras transplantadas musculares

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

In vivo macrófagos Imagem Usando MR alvejado agente de contraste para avaliação longitudinal de artrite séptica

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

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

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

Um Modelo murino de próstata humana Metástase Ortotópico

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

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Contraste Dinâmico aprimorada Ressonância Magnética de um Modelo de câncer de pâncreas Orthotopic Rato

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Geração de paciente com câncer de próstata Derivado modelos de xenotransplante de células tumorais circulantes

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Um tumor cerebral / Slice organotípicas Co-cultura Sistema de Estudos de Tumor Microenvironment e alvejado droga Terapias

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...