RESUMO
The use of digital pathology for the histomorphologic profiling of pathological specimens is expanding the precision and specificity of quantitative tissue analysis at an unprecedented scale; thus, enabling the discovery of new and functionally relevant histological features of both predictive and prognostic significance. In this study, we apply quantitative automated image processing and computational methods to profile the subcellular distribution of the multi-functional transcriptional regulator, Kaiso (ZBTB33), in the tumors of a large racially diverse breast cancer cohort from a designated health disparities region in the United States. Multiplex multivariate analysis of the association of Kaiso's subcellular distribution with other breast cancer biomarkers reveals novel functional and predictive linkages between Kaiso and the autophagy-related proteins, LC3A/B, that are associated with features of the tumor immune microenvironment, survival, and race. These findings identify effective modalities of Kaiso biomarker assessment and uncover unanticipated insights into Kaiso's role in breast cancer progression.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Transcrição/metabolismo , Microambiente Tumoral , Automação Laboratorial , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Interpretação de Imagem Assistida por Computador , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Prognóstico , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Transdução de Sinais , Fatores de Tempo , Análise Serial de Tecidos , Fatores de Transcrição/genética , Evasão Tumoral , Estados Unidos/epidemiologiaRESUMO
Multiplexed immunohistochemistry (mIHC) enables the detection, quantification, and localization of many markers within cell or tissue samples, leading to a better understanding of the architecture of a disease at the cellular level. Current mIHC techniques involve long staining and assay times, require dedicated and/or captive instrumentation, and entail tedious assay optimization, hindering their establishment as routine methods. Here, we demonstrate the use of the InSituPlex® method for spatial profiling of immuno-oncology targets in FFPE tumor tissue with the UltiMapper™ I/O PD-L1 multiplex assay. The panel consists of five protein markers to profile immune infiltration and PD-L1 expression and includes CD8, CD68, PD-L1, pan CK, and SOX10 markers. The assay shows benefits of high and low expression of markers, coexpression and colocalization of proteins in single cells, and completion of staining and image acquisition in 5.5 h. Through the combination of multiplexed characterization of protein expression in whole tissue sections, fast staining workflow, and compatibility with existing instrumentation, the InSituPlex method provides a robust modality for deep phenotyping of the tumor and its microenvironment.
Assuntos
Biomarcadores Tumorais/metabolismo , Perfilação da Expressão Gênica/métodos , Neoplasias/metabolismo , Coloração e Rotulagem/métodos , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno B7-H1/metabolismo , Antígenos CD8/metabolismo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Inclusão em Parafina , Fenótipo , Fatores de Transcrição SOXE/metabolismo , Análise Espacial , Fixação de TecidosRESUMO
The detection of proteins in 2-D gels and their subsequent identification by MS is still the "gold standard" in proteomics. Fluorescent detection has increasingly replaced colorimetric and radiometric detection on gels and blots. The reasons for this are multiple and varied and include higher sensitivity, better quantitation, increased dynamic range, speed, safety and ease of use. Unlike other methods, fluorescent protein detection is also typically very consistent in response from protein to protein and in many cases is compatible with MS methods for protein identification. The superior sensitivity and benefits achieved by fluorescent techniques have spurred the development of instrumentation capable of delivering precise, sensitive, high-resolution image acquisition over a wide variety of excitation and emission wavelengths. This report focuses on applications using the highly sensitive, charge-coupled device based ProXPRESS multilabel imager, readily configurable for image acquisition over a wide variety of wavelengths (380-700 nm and ultraviolet (UV)) using xenon lamp or UV excitation. The ability to simultaneously detect enzyme activities or protein modifications with different color fluorescent probes in addition to total protein amounts (multiplexing) allows the further mining of proteomic data content from a single set of protein samples. To this end, the development of instrumentation that enables a multiplexing strategy will become central to in-depth proteomic studies. The ProXPRESS maximizes the efficiency of experimental strategies that require flexibility and multicolor fluorescence detection.