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1.
J Immunol Methods ; 478: 112714, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31783023

RESUMO

With the explosion of immuno-oncology and the approval of many immune checkpoint therapies by regulatory agencies in the last few years, understanding the tumor microenvironment (TME) in the context of patients' immune status has become essential. Among available immune profiling techniques, multiplex immunofluorescence (mIF) assays offer the unique advantage of preserving the architectural features of the tumor and revealing the spatial relationships between tumor cells and immune cells. A number of mIF and image analysis assays have been described for solid tumors but most are not sufficiently suitable in lymphoma, where the lack of clear tumor-stromal boundaries and high tumor density present significant challenges. Here we describe the development and optimization of a reliable workflow using Akoya Opal staining kits to label and analyze 6 markers per slide in diffuse large B-cell lymphoma (DLBCL) tissue sections. Five panels totaling 30 markers were developed to characterize infiltrating immune cells and relevant check-point proteins such as PD1, PD-L1, ICOS, SIRP-alpha and Lag3 on 70 DLBCL sections. Multiplexed sections were scanned using an Akoya multispectral scanner. An image analysis workflow using InForm and Matlab was developed to overcome challenges inherent to the DLBCL environment. Using the assays and workflows detailed here, we were able to quantify cell densities of subsets of infiltrating immune cells and observe their spatial patterns within the tumors. We highlight heterogeneous distribution of cytotoxic T cells across tumors with similar T cell density to underscores the importance of considering spatial context when studying the effects of immunological therapies in DLBCL.


Assuntos
Biomarcadores Tumorais/análise , Imunofluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Linfoma Difuso de Grandes Células B/imunologia , Microambiente Tumoral/imunologia , Algoritmos , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Estudos de Viabilidade , Imunofluorescência/instrumentação , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Processamento de Imagem Assistida por Computador , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Reprodutibilidade dos Testes , Software , Análise Espacial , Coloração e Rotulagem , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Fluxo de Trabalho
2.
Nat Struct Mol Biol ; 21(9): 803-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25108355

RESUMO

The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.


Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas de Ligação a DNA/metabolismo , Peptídeo Hidrolases/metabolismo , Talidomida/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Animais , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Humanos , Lenalidomida , Camundongos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Talidomida/química , Talidomida/farmacologia , Ubiquitina-Proteína Ligases
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