Name: quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections
Uploaded: 2017-07-31T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Os autores agradecem os Drs. Fahad Bafakih e Jim Mandell para aquisição e identificação de amostras de pacientes, Garrett F. Beeghly para assistência com imuno-histoquímica e o Centro de Pesquisa de Biorepositórios e Tecidos, Centro de Pesquisa Cardiovascular Center Histology Core e o Centro de Análise Biomolecular da Universidade da Virgínia para assistência com Aquisição de amostras, imuno-histoquímica e imagens.

Este protocolo identifica componentes celulares em amostras embutidas em parafina fixada em formalina (FFPE), como é típico para amostras de pacientes clínicas bancárias. A incorporação de parafina permite a melhor manutenção da morfologia celular e tecidual, além de ter uma melhor longevidade das seções. As amostras utilizadas para esta análise foram acessadas através da Universidade de Virginia Biorepository e Tissue Research Facility. As amostras de pacientes foram selecionadas por um neuropatologista com base em um diagnóstico definitivo de glioblastoma (astrocitoma, grau IV da OMS) que completaram ressecções tumorais na Universidade da Virgínia entre 2010 e 2013 e foram des-identificados antes dessa análise 11 . 1. Desparaffinização e Reidratação de Amostras FFPE NOTA: Esta parte do protocolo é específica para amostras FFPE. Embora as amostras embutidas em parafina possam ser mais úteis para esta análise, devido à preservação de células e tQuestão de morfologia, esta análise também pode ser feita com seções congeladas. Se estiver usando seções congeladas, esta porção pode ser omitida e proceder diretamente à imuno-histoquímica cromogênica. <ol><li> Execute as seguintes lavagens por 5 minutos cada: Xileno, Xileno, 100% Etanol, 100% Etanol, 95% Etanol, 95% Etanol, 70% Etanol, Água desionizada, Água desionizada </li></ol> 2. Antigen Retrieval NOTA: Esta porção do protocolo é necessária para quebrar pontes de metileno formadas durante a fixação de formalina de amostras de FFPE e expor sites de antígenos para anticorpos para se ligar. <ol><li> Diluir a solução de desmascaramento de antigénio de alto pH com base em Tris na recomendação do fabricante em água destilada. </li><li> Execute a recuperação do antígeno mediada pelo calor usando um microondas. Outras formas de recuperação mediada pelo calor (como panela de pressão, vaporizador de vegetais ou água fervente) também seriam suficientes. <ol><li> Adicione a solução de desmascaramento diluída a um microondas não seladoembarcação. Coloque as lâminas na embarcação. Coloque as lâminas no microondas. </li><li> Ferver durante 20 minutos com alta potência. Monitore os níveis de líquido para evaporação e reabasteça com água destilada conforme necessário. </li></ol></li><li> Permitir que as amostras esfriem em solução durante 1 h à temperatura ambiente. </li></ol> 3. Imunohistoquímica cromogênica <ol><li> Esboce uma amostra de tecido com uma caneta hidrofóbica para minimizar o volume de reagentes necessários para cobrir a amostra. NOTA: Certifique-se de manter as amostras de tecido hidratadas e não as deixe secar, pois isso afetará a eficácia da coloração. </li><li> Pipetar uma solução de permeabilização suficiente (solução salina tamponada com Tris (TBS) + Triton-X a 0,01% para cobrir a amostra de tecido (tipicamente cerca de 100 a 200 μL). </li><li> Remova e descarte a solução e repita o passo 3.2. </li><li> Incubar amostras à temperatura ambiente com solução de bloqueio (2,5% de soro de cavalo + solução de permeabilização). </li><li> Incubar amostras durante a noite em 4 ºC com anticorpos primários diluídosEm solução de bloqueio. NOTA: Todos os anticorpos primários utilizados aqui são diluídos a 1: 200, mas as diluições ideais podem ser determinadas usando diluições em série, começando com a recomendação do fabricante. Informações detalhadas sobre os anticorpos usados ​​neste protocolo foram previamente publicadas 11 . </li><li> Detectar anticorpos primários usando um reagente de polímero de peroxidase de rábano correspondente ao animal hospedeiro do anticorpo primário, seguindo o protocolo do fabricante. </li><li> Pipetar solução de permeabilização suficiente para cobrir a amostra de tecido e incubar durante 5 min. </li><li> Remova e descarte a solução e repita a Etapa 3.7. </li><li> Incubar as lâminas em 0,3% de H 2 O 2 em 1x TBS durante 15 min. </li><li> Desenvolva amostras com um substrato de peroxidase diaminobenzidina (DAB) durante 2 a 10 min até atingir a intensidade de mancha desejada. </li><li> Contra-amostras para identificar núcleos celulares, como com hematoxilina, seguindo o protocolo do fabricante. </li><li> Desidratar amostras com 100%Etanol e xileno. </li><li> Monte amostras permanentemente com mídia de montagem. </li></ol> 4. Identificação das Regiões de Interesse <ol><li> Lâminas de imagem sob microscópio de campo brilhante capaz de imagens de alta resolução. NOTA: use componentes celulares de imagem com uma resolução mínima de 20x. </li><li> Mova a câmera para regiões específicas de interesse em amostras de tecido. </li><li> Salve imagens como arquivos TIFF para quantificação. </li></ol> 5. Análise de imagem <ol><li> Abra imagens no ImageJ para quantificar a cobertura percentual. </li><li> Use o plug-in Limite de cor para remover a cor roxa dos núcleos corados com hematoxilina. </li><li> Converta a imagem para 8 bits. </li><li> Adicione um limite sem fundo escuro. </li><li> Processe a imagem usando um dos 17 filtros de limiar ImageJ pré-carregados ( ou seja, MaxEntropy), de modo que apenas as porções coloridas DAB estão incluídas no limite. NOTA: Selecione o filtro de limite pré-carregado ótimo que minimizouÉ inclusão de coloração de fundo. Isso pode depender da qualidade da coloração e especificidade do anticorpo. Use o mesmo limite para todas as repetições técnicas dentro de cada amostra de paciente. </li><li> Aplicar limite. </li><li> Medir a área de porcentagem da imagem limite. </li><li> Cobertura de área percentual média para múltiplas regiões dentro de cada amostra. </li></ol>

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Nosso método aqui proposto é uma abordagem quantitativa para analisar amostras histológicas coradas usando imunohistoquímica cromogênica tradicional. A metodologia atual para este tipo de análise inclui protocolos de coloração semelhantes, seguidos pela classificação por patologistas independentes. Este método tem sido confiável, no entanto, para uma série de aplicações, é necessária uma compreensão mais precisa da maquiagem celular, como melhor compreensão da heterogeneidade associada aos tumores e r...

O glioblastoma (GBM), o câncer cerebral mais comum e maligno, é caracterizado por invasão altamente difusa do tumor primário para o parênquima 1 , 2 do cérebro circundante. Essa invasão difusa torna o tumor particularmente difícil de ressecar completamente, e as células cancerígenas invasoras que permanecem pós-terapia são a razão mais comum para recorrência inevitável 2 , 3 , 4 . Anteriormente, achamos inibir a invasão de células de glioma difuso para ser terapeuticamente benéfico 5 , porém pouco se sabe sobre os mecanismos complexos que contribuem para a invasão GBM. O microambiente do tumor, ou tecido que envolve o câncer, tem sido implicado na progressão de tumores em cânceres múltiplos 6 , 7 . O microambiente tumoral do glioblastoma, em pArticular, é relativamente pouco caracterizada e é exclusivamente complexa, composta de células gliais múltiplas, como astrocitos, microglia e oligodendrócitos, bem como matriz extracelular, fatores solúveis e fatores biofísicos. Experimentalmente, os astrocitos e a microglia demonstraram aumentar a progressão do glioma e a invasão 8 , 9 , 10 , mas a composição de todas as células gliais no microambiente do cérebro humano nativo é desconhecida. Nós anteriormente mostramos componentes microambientais que podem prever a sobrevivência do paciente analisando quantitativamente os componentes celulares do microambiente do glioblastoma e incorporando nossas análises em um modelo de riscos proporcionais 11 . Aqui, descrevemos o método de análise quantitativa para identificar as populações de células gliais dentro do tumor a granel e regiões adjacentes de ressecções tumorais do paciente com glioblastomaS. Utilizamos imuno-histoquímica cromogênica para identificar as populações de células gliais e ImageJ para analisar a cobertura percentual de coloração para cada população glial. Avaliar a cobertura percentual cria uma medida simples para determinar as diferenças morfológicas das células, particularmente aquelas afetadas pelas interações com células cancerígenas. Estudos anteriores para quantificar a coloração histopatológica usam coloração padrão, como hematoxilina e eosina 12 ou tricromo 13 de Masson, que não aproveitam a especificidade da coloração imuno-histoquimica baseada em anticorpos. Nosso método foi desenvolvido para quantificar diretamente as populações gliais dentro das resecções tumorais do paciente com glioblastoma, que pretendemos usar para elucidar o microambiente do glioblastoma complexo.

<table><tbody><tr><td>Xylene</td><td>Fisher Chemical</td><td>X3P</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>High pH antigen unmasking solution</td><td>Vector Labs</td><td>H-3301</td><td></td></tr><tr><td>TBS</td><td></td><td></td><td></td></tr><tr><td>Triton-X</td><td>Amresco</td><td>9002-93-1</td><td></td></tr><tr><td>Horse serum</td><td></td><td></td><td></td></tr><tr><td>Anti-ALDH1L1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab56777</td><td></td></tr><tr><td>Anti-Iba1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab5076</td><td></td></tr><tr><td>Anti-Oligodendrocyte Specific Protein1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab53041</td><td></td></tr><tr><td>ImmPRESS anti-goat</td><td>Vector Labs</td><td>MP-7405</td><td></td></tr><tr><td>ImmPRESS Universal (anti-mouse/rabbit)</td><td>Vector Labs</td><td>MP-7500</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma Aldrich</td><td>216763</td><td></td></tr><tr><td>ImmPACT DAB substrate</td><td>Vector Labs</td><td>SK-4105</td><td></td></tr><tr><td>Hematoxylin counterstain</td><td>ThermoScientific</td><td>72404</td><td></td></tr><tr><td>Histochoice Mounting Media</td><td>Amresco</td><td>H157-475</td><td></td></tr><tr><td>Aperio Scanscope</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Image Scanscope</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Super HT PAP Pen</td><td>Research Products International</td><td>195506</td><td></td></tr></tbody></table>

quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections

Com o crescente interesse no microambismo do tumor, buscamos desenvolver um método para determinar especificamente os componentes do microambiente dentro de amostras de pacientes com glioblastoma, o câncer cerebral mais mortal e invasivo. Não só os métodos quantitativos são benéficos para descrever com precisão os tecidos doentes, mas também podem contribuir para um prognóstico mais preciso, diagnóstico e desenvolvimento de sistemas e substituições de engenharia de tecidos. No glioblastoma, as células gliais, como a microglia e os astrócitos, foram correlacionadas de forma independente com um mau prognóstico com base na classificação de patologistas. No entanto, o estado dessas células e outros componentes das células gliais não foram bem descritos de forma quantitativa. Isso pode ser difícil devido aos grandes processos que marcam essas células gliais. Além disso, a maioria das análises histológicas se concentra na amostra de tecido geral ou apenas na maior parte do tumor, ao contrário de delinear as quantificações com base em regiõesSobre o tecido altamente heterogêneo. Aqui, descrevemos um método para identificar e analisar quantitativamente as populações de células gliais dentro do tumor a granel e regiões adjacentes de ressecções tumorais de pacientes com glioblastoma. Utilizamos imuno-histoquímica cromogênica para identificar as populações de células gliais em resecções tumorais do paciente e ImageJ para analisar a cobertura percentual de coloração para cada população glial. Com essas técnicas, podemos descrever melhor as células gliais em todas as regiões do microambiente do tumor de glioma.

Para esta análise, duas regiões de interesses dentro de nossas ressecções tumorais - o tumor primário e as regiões adjacentes, principalmente composta de tecido saudável com células invadidas de câncer invasivo ( Figura 1A , 1B ) - foram identificadas por neuropatologistas colaboradores em hematóxilina e paciente com corante de eosina Amostras. Dentro de cada amostra de paciente, identificou-se a coloração positiva para astrocito...

Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Imuno-histoquímica quantitativa do microambiente celular em resecações do glioblastoma do paciente

Este protocolo foi desenvolvido para identificar quantitativamente os componentes do microambiente tumoral em resecções do paciente com glioblastoma usando imuno-histoquímica cromogênica e ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

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9.6K visualizações.  University of Virginia.  Este protocolo foi desenvolvido para identificar quantitativamente os componentes do microambiente tumoral em resecções do paciente com glioblastoma usando imuno-histoquímica cromogênica e ImageJ. 

Vídeo: Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Co-cultura Ensaios para estudar macrófagos e Microglia Estimulação do Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Pesquisa do câncer

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

Enriquecimento e caracterização do microambiente que imune e não imune Tumor em tumores murino subcutâneos estabelecidas

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Avaliação de biomarcadores em Glioma por imuno-histoquímica na parafina Glioma 3D Neurosphere culturas

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Otimização da cultura celular glioma de alto grau a partir de espécimes cirúrgicos para uso em modelos animais clinicamente relevantes e imunoquímica 3D

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Medicina

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Perfil Espacial Digital para Caracterização do Microambiente em Glioma Infiltrante Difuso do Tipo Adulto

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

Imunofluorescência Multiplex e Análise de Imagem Espacial do Microambiente Tumoral

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Autor em destaque: Imunofluorescência multiplex combinada com análise de imagens espaciais para a avaliação clínica e biológica do microambiente tumoral  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

Imunofluorescência Multiplex Combinada com Análise Espacial de Imagens para Avaliação Clínica e Biológica do Microambiente Tumoral

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolamento e Análise de Citometria de Fluxo infiltrantes Glioma Células Mononucleares do Sangue Periférico

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Imunologia e Infecção

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Estereotáxica transferência adotiva de citotóxicas células imunes em modelos murino de humanos ortotópico Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Imuno-histoquímica quantitativa do microambiente celular em resecações do glioblastoma do paciente

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