Esta pesquisa foi suportada pelo Caso Comprehensive Cancer Center (P30 CA43703).

1. Estereotáxica Cyberknife Consulta Radiocirurgia <ol><li> Descrever Cyberknife tratamento radiocirurgia. Cyberknife radiocirurgia envolve o uso de um acelerador linear montado sobre um braço robótico industrial semelhante às máquinas utilizadas para fazer automóveis. O braço robótico se move o acelerador linear no espaço tridimensional em torno de um paciente à medida que progride através de &quot;pára&quot; tratamento múltiplo para entrega do feixe. Cruz-plano raios-x são obtidos durante o tratamento para verificar o paciente está em uma posição de tratamento correcto. Uma vez que a entrega de feixe de tratamento não é restrita a um plano bidimensional, este sistema melhora ainda mais a capacidade de entregar doses de radiação concentradas para alvos cancerosas enquanto minimiza a dose de radiação para a pele crítica e de estruturas de órgãos viscerais. </li><li> Discutir os riscos radiocirurgia CyberKnife. Cyberknife radiocirurgia pode resultar em possível curtimento e vermelhidão da pele, náuseas, fadiga infreqüente ou diarrhea, lesões de órgãos rara visceral, músculo, nervo e lesão óssea e risco muito pequeno de segundo câncer. </li></ol> 2. Radiocirurgia estereotáxica Cyberknife Colocação Fiducial <ol><li> Descreva colocação fiducial. Mulheres em tratamento para câncer ginecológico persistentes ou recorrentes sofrem operatório ou CT-guiada colocação de pelo menos três simples 1,6 x 3 mm de ouro fiducials tecido mole (aproximadamente do tamanho de um grão de arroz) dentro ou perto do alvo câncer. Clipes cirúrgicos colocados no momento da cirurgia, antes não são de densidade suficiente para ser usado como fiduciais radiocirúrgico. Clipes cirúrgicos não interfiram com radiocirúrgico segmentação por causa da discrepância densidade. </li><li> Indique posicionamento fiducial em relação ao alvo radiocirúrgico. Fiducials são colocados em diferentes profundidades de tecidos ao redor do alvo radiocirúrgico e devem ser separados por dois centímetros ou mais. Fiducials estão posicionados dentro de 4 a 6 centímetros do target. </li></ol> 3. Estereotáxica Cyberknife Plano de Tratamento Radiocirurgia <ol><li> Discutir o planejamento do tratamento radiocirúrgico. Pelo menos uma semana após a colocação fiducial (ou seja, para que a cura tenha ocorrido e movimento fiducial é minimizado), os pacientes passam CT-guiada simulação tratamento SBRT. Em nosso programa, de dois pinos localizados evacuado imobilização vácuo saco-pélvica é usado. </li><li> Indique o posicionamento do paciente na mesa de tratamento. As mulheres são tratadas em uma posição tratamento supina sobre a mesa radiocirúrgico Cyberknife plana. </li><li> Discuta-alvo e imobilização do paciente. Paciente pode sofrer imobilização evacuados vácuo saco para reduzir o movimento intrafraction durante o parto dose de radiação. Uma vez imobilizado, as mulheres sofrem não-contrastada contígua tomografia axial computadorizada de alta resolução de imagem (1 a 1 passo, 1,0 espessura de corte mm, tensão de 120 kVp, 450 mAs). </li><li> Discutir o uso de avançadaimagem para a localização alvo. Após tomografia computadorizada, as mulheres sofrem de preferência convencional 18 F-FDG PET / CT na mesma posição SBRT tratamento. Alternativamente, as mulheres podem ser submetidos a convencional contrastado ressonância magnética de pelve. </li><li> Descreva imagem co-registo para o planejamento do tratamento radiocirúrgico. Imagens de TC de alta resolução e 18 F-FDG PET / CT são importados para o planejamento inverso radioterapia no Sistema de Planejamento MultiPlan 3.5.2 Tratamento (Accuray). </li><li> Descreve a abordagem da equipe para o planejamento do tratamento radiocirúrgico. Para o planejamento do tratamento, tanto oncologista uma radiação e oncologista ginecológico câncer de contorno alvo volumes-alvo clínicos (TVC). Outros doença do tecido em risco é contornado e está incluído na CTV. Perto estruturas de tecidos normais, como o intestino delgado, reto, bexiga, fígado, rins, pulmões, fêmur proximal bilaterais, vagina, e as raízes nervosas sacrais são contornadas poro oncologista. </li><li> Descrever prescrição dose de radiação. Uma dose de radiação de prescrição 3 × 800 cGy = 2400 cGy (vulgarmente para a linha de isodose 70%) foi seleccionada. Em um cálculo da dose de 200 cGy biológico equivalente, cerca de 6.170 cGy são entregues com esta receita assumindo um &quot;α / β&quot; proporção de 10 por tumor. A radiação emitida a partir de um acelerador linear SBRT é colimada, quer usando um de 12 colimadores de tungsténio fixos circulares (5 a 60 mm) ou usando um tungsténio-cobre liga segmentado hexágono colimador IRIS. 14 órgãos normais têm uma tolerância de radiação e de utilizar parâmetros de dose-volume do histograma para restrição tecido normal durante radiocirurgia ablativo estão listados na Tabela 1. </li></ol> 4. Estereotáxica Cyberknife Entrega Tratamento Radiocirurgia <ol><li> Execute cruz plano de verificação de imagem. Durante Cyberknife SBRT, tecidos moles fiduciais(Ou em alguns casos rígidos marcos anatômicos, como o crânio de lesões intracranianas e da coluna vertebral para as lesões da coluna vertebral) são controladas por cruz plano radiografia e confirmou estar no local de destino previsto pelo sistema de destino localização (TLS) subsistema. </li><li> Verificar posicionamento alvo. Fiducials são monitorados em três e até seis graus de liberdade (ou seja, x, y, z, pitch, roll e yaw). As imagens geradas pelos TLS são automaticamente registradas e comparadas com radiografias reconstruídas digitalmente (DRRs) gerados a partir do tratamento inicial planejando tomografia computadorizada. </li><li> Verificar o posicionamento alvo. Se os resultados do registo automático indicam que fiduciais ter deslocado para além tolerâncias pré-definidos, de qualquer um dos seis graus de liberdade (x, y, z, breu, rolo, e de guinada), o tratamento é automaticamente pausa. Reposicionamento do paciente, em seguida, ocorre. </li><li> Considere o movimento alvo. Metas que se movem com movimento respiratório são treated utilizando o Synchrony subsistema de Controle Respiratório (sincronia). Com a utilização de uma câmara montada na sala de tratamento, o sistema rastreia continuamente padrão respiratório do paciente (isto é, obter os dados de posição externas) com a utilização de diodos emissores de luz (LEDs) afixados a um colete em torno do tórax do paciente. 10 Juntos com as informações de localização fiducial (dados de posição internas) obtido a partir de cruzamento de plano de raios-X, o sistema Synchrony constrói um modelo de correlação com os dados externos e os dados internos. Este modelo de correlação permite que o braço robótico para acompanhar qualquer movimento respiratório induzida do alvo, enquanto durante a entrega de toda e qualquer feixes de tratamento. </li><li> O tratamento pode implicar 100-150 posições tratamento liberados do braço robótico. Os tratamentos podem durar de 30 a 90 minutos. </li></ol> 5. Os resultados representativos Radioterapia estereotáxica corpo (SBRT) pode envolver muitos feixes de radiação individuais (<html> vectores azuis) Todos os convergindo para simples ou múltiplas, estreitamente associados alvos de radiação clínica, como mostrado na Figura 1A. Um resultado de planejamento bom representante radiocirúrgico dá um tratamento SBRT entrega com alta dose de radiação cobertura câncer volume alvo e conformalidade alvo câncer. Figuras 1B-D mostra 131 vigas foram usados ​​para tratar sobre uma recaída pélvica de um alvo chemorefractory câncer de ovário mais de 42 minutos. SBRT prescrito para a linha de isodose 80% processada 100% de cobertura de volume clínica alvo com um índice de conformalidade de 1,94 para uma dose total de 2400 cGy em três fracções diárias de 800 cGy. Os histogramas de dose-volume para o alvo clínico (vermelho) e para as estruturas críticos de recto (castanho), bexiga (amarelo), intestino delgado (luz azul) sacral nervos (TAN) e quadris (laranja) estão representados na Figura 2. <img alt="A Figura 1" src="/files/ftp_upload/3793/3793fig1.jpg" /> Figura 1.tp :/ / www.jove.com/files/ftp_upload/3793/3793fig1large.jpg &quot;target =&quot; _blank &quot;&gt; Clique aqui para ver maior figura. <img alt="A Figura 2" src="/files/ftp_upload/3793/3793fig2.jpg" /> Figura 2. Histogramas dose-volume para o alvo clínico (vermelho) e para as estruturas críticas do reto (marrom), bexiga (amarela), intestino delgado (luz azul) sacrais nervos (TAN) e quadris (laranja).

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Nós (CAK, JMB, RD) não têm nada a revelar.

Incentivar os resultados SBRT primeiros têm alimentado a investigação clínica da radiocirurgia para tratamento de persistentes ou recorrentes cânceres ginecológicos. 7, 8, 15 Dados questionar efeitos radiobiológicos e modo de morte celular resultante da SBRT. Pequenos estudos clínicos demonstraram que as doses de radiação ablativos fornecidos pelo SBRT produzir taxas de doenças específicas de controle superiores a 90%. Ao contrário com radiação convencional, tem sido um desafio para combinar SBRT com quimioterapias radiosensíveis e citotóxico / citostático. Melhor câncer alvo através de um aumento de planejamento expansões de volume de tratamento e através da inclusão de 18 F-FDG PET / CT imagens melhorou os resultados clínicos. Enquanto é imperativo olhar para outros métodos de entrega de dose-escalada de radiação com alta precisão, ele permanece indeciso se SBRT pode proporcionar uma eficácia terapêutica equivalente como de baixa e alta taxa de dose de braquiterapia. De facto, a braquiterapia é a mais comum e validatado técnica para conseguir o aumento da dose de radiação em alvos de câncer ginecológicos. Como tal, tanto entusiasmo e prudência são apropriados em leitura de dados SBRT disponíveis para o tratamento dos cânceres ginecológicos.

<table border="1"><tbody><tr><td> Nome do dispositivo </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Cyberknife sistema </td><td> Accuray (Sunnyvale, CA) </td><td></td><td></td></tr></tbody></table>

stereotactic radiosurgery for gynecologic cancer

Radioterapia estereotáxica corpo (SBRT) distingue-se pela necessitando mais rígida imobilização do paciente, representando o movimento respiratório, plano de tratamento complexo, a bordo de imagem, e número reduzido de doses de radiação ablativos para o câncer atinge geralmente refratário à quimioterapia e radioterapia convencional. Campos dose de radiação íngreme SBRT drop-off autorizações estreito &#39;feixe lápis&#39; de tratamento a ser utilizado para o tratamento de radiação ablativa condensada em 1 a 3 tratamentos. Tratar os médicos devem perceber que SBRT tem um maior perigo de lesão do tecido normal e chance de perder tumor geográfica. Ambos devem ser enfrentados pela imobilização de metas de câncer e de alta precisão de entrega tratamento. Imobilização alvo câncer foi conseguido através do uso de moldes personalizados indexados de isopor, sacos de feijão evacuados, ou corpo-fix moldes com paciente independente de compressão abdominal. Movimento Intrafraction 1-3 de tarja câncerts devido a respirar agora pode ser reduzida por paciente-responsivas técnicas de respiração preensão, 4 paciente bocal de coordenação de respiração activa, 5 respiração-correlacionado tomografia computadorizada, 6 ou guiada por imagem de rastreamento de fiduciais implantados no interior e em torno de um tumor em movimento. 7-9 A Cyberknife sistema (Accuray [Sunnyvale, CA]) utiliza um acelerador linear radiação montado sobre um braço robótico industrial que segue o movimento com precisão respiratório do paciente por uma câmara-rastreadas conjunto de diodos emissores de luz (LED) impregnadas em um colete equipado a um paciente. 10 reduções substanciais nas margens de terapia por radiação pode ser conseguido por rastreamento de movimento, em última análise, processamento de um menor volume de planeamento de destino que são irradiados com precisão submillimeter. 11-13 Metas de câncer tratados pelo SBRT são irradiados por feixes convergentes, bem colimados. Dose de radiação resultante de histogramas de câncer de volume-alvo têm um pronome maisced radiação &quot;ombro&quot;, indicando alta porcentagem de cobertura alvo e uma pequena alta dose de radiação &quot;cauda&quot;. Assim, conformalidade alvo aumento vem à custa de uniformidade de dose diminuiu no alvo cancro SBRT. Isto pode ter implicações tanto para o controle do tumor subseqüente no alvo SBRT e tolerância do tecido normal dos órgãos de risco. Devido à queda acentuada na dose de SBRT, a possibilidade de doença oculta escapar dose de radiação ablativo ocorre quando alvos de câncer não são totalmente reconhecidos e margens de doses inadequadas SBRT são aplicadas. Clínica alvo volume de expansão (CTV) por 0,5 cm, resultando em um volume maior de planeamento alvo (PTV), está associada com controle de destino aumentado sem lesão tecidual indevida normal. 7,8 redução adicional na probabilidade de erro geográfico pode ser conseguida por incorporação de 2 - [18 F]-fluoro-2-desoxi-D-glucose (18 F-FDG) tomografia por emissão de positrões (PET) 8 Utilização de 18 F-FDG PET / CT em SBRT treatme.planejamento nt é apenas o começo de tentativas para descobrir novas assinaturas de imagem alvo molecular para cânceres ginecológicos.

Watch this Scientific Journal Video about Stereotactic Radiosurgery for Gynecologic Cancer at JoVE.com

Stereotactic Radiosurgery for Gynecologic Cancer

Radioterapia estereotáxica corpo (SBRT) envolve guiada por imagem de radiação, ablativo entregue ao câncer tem como alvo refratário à quimioterapia ou ao tratamento de radiação convencional. O robô armado Cyberknife sistema SBRT, usando localização alvo sofisticado, oferece doses de radiação hipofracionada capazes de esterilizar alvos de câncer. Este artigo irá considerar novas funções terapêuticas da SBRT para cânceres ginecológicos.

Radiocirurgia Estereotáxica para Câncer Ginecológico

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, ...

stereotactic-radiosurgery-for-gynecologic-cancer

Research

JoVE Journal

Medicine

17.5K visualizações.  University Hospitals Case Medical Center and Case Western Reserve University School of Medicine. Radioterapia estereotáxica corpo (SBRT) envolve guiada por imagem de radiação, ablativo entregue ao câncer tem como alvo refratário à quimioterapia ou ao tratamento de radiação convencional. O robô armado Cyberknife sistema SBRT, usando localização alvo sofisticado, oferece doses de radiação hipofracionada capazes de esterilizar alvos de câncer. Este artigo irá considerar novas funções terapêuticas da SBRT para cânceres ginecológicos.

Vídeo: Stereotactic Radiosurgery for Gynecologic Cancer

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Imagens em tempo real multiespectrais de fluorescência para detecção intra-operatória do linfonodo sentinela em Oncologia Ginecológica

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Medicina

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Implantação estereotáxica intracraniana e<em&gt; Em vivo</em&gt; Imaging Bioluminescent de xenoenxertos de tumores em um Sistema Modelo de Rato Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

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. ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Modelo ortotópico bioluminescente de progressão do câncer de pâncreas

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling de MicroRNAs estrogênio regulados em células do cancro da mama

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

Utilizando Murino Inducible telomerase alelos nos Estudos de degeneração / regeneração tecidual e Câncer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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 ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Trabalhando com tecidos humanos para Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modelando os passos iniciais do cancro do ovário Divulgação em uma cultura organotípicas do Peritoneal Cavity Humano

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...