Multiplex Immunofluorescence Assays

Francisco‐Cruz, Alejandro; Parra, Edwin R.; Tetzlaff, Michael T.; Wistuba, Ignacio I.

doi:10.1007/978-1-4939-9773-2_22

Cited by 54 publications

(46 citation statements)

References 59 publications

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“…Human tonsil FFPE tissues were included and used as positive and negative (autofluorescence) control with the primary antibodies plus fluorophores and with primary antibodies without fluorophores, respectively. The stained slides were scanned using Vectra 3.0 multispectral microscope system (Akoya Biosciences, USA) under fluorescent illumination as previously described 71 , first in low magnification (×10), then the representative regions of interest (ROIs) were selected with Phenochart1.0.9 viewer (Akoya Biosciences, USA) and finally scanned in high magnification (×20). The ROIs (669 × 500 μm each) for mIF analysis were then selected after comparing with H&E slides to capture malignant and premalignant cell cluster and various elements of heterogeneity.…”

Section: Methodsmentioning

confidence: 99%

Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features

Dejima

Chen

et al. 2021

Nat Commun

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The mechanism by which anti-cancer immunity shapes early carcinogenesis of lung adenocarcinoma (ADC) is unknown. In this study, we characterize the immune contexture of invasive lung ADC and its precursors by transcriptomic immune profiling, T cell receptor (TCR) sequencing and multiplex immunofluorescence (mIF). Our results demonstrate that anti-tumor immunity evolved as a continuum from lung preneoplasia, to preinvasive ADC, minimally-invasive ADC and frankly invasive lung ADC with a gradually less effective and more intensively regulated immune response including down-regulation of immune-activation pathways, up-regulation of immunosuppressive pathways, lower infiltration of cytotoxic T cells (CTLs) and anti-tumor helper T cells (Th), higher infiltration of regulatory T cells (Tregs), decreased T cell clonality, and lower frequencies of top T cell clones in later-stages. Driver mutations, chromosomal copy number aberrations (CNAs) and aberrant DNA methylation may collectively impinge host immune responses and facilitate immune evasion, promoting the outgrowth of fit subclones in preneoplasia into dominant clones in invasive ADC.

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Section: Methodsmentioning

confidence: 99%

Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features

Dejima

Chen

et al. 2021

Nat Commun

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“…The stained slides were scanned with Vectra 3.0 multispectral microscope system (Akoya Biosciences, USA) under fluorescent illumination. These mIF assays followed our previous publication (Francisco-Cruz et al, 2020).…”

Section: Star Methodsmentioning

confidence: 99%

Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features

Dejima

Chen

et al. 2020

Preprint

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The evolution of anti-tumor immune surveillance during initiation and progression of preneoplasia into invasive lung adenocarcinoma (ADC) and its underlying molecular changes are largely unknown. To fill this void, we characterized the immune contexture of invasive lung ADC (n=12) and its precursors of consecutive developmental stages including preneoplasia atypical adenomatous hyperplasia (AAH, n=22), adenocarcinoma in situ (AIS, n=16), minimally invasive adenocarcinoma (MIA, n=28) as well as paired normal lung tissues (NL, n=53) by transcriptomic profiling of 770 genes of nCounter PanCancer Immune Profiling Panel (Nanostring), T cell receptor (TCR) sequencing and multiplex immunofluorescence (mIF). Our results demonstrated that anti-tumor immunity evolved as a continuum from AAH to AIS, MIA and invasive lung ADC with a gradually less effective and more intensely regulated immune response evidenced by down-regulation of immune activation pathways, up-regulation of immunosuppressive pathways, higher infiltration of CD4+ T cells, lower infiltration of CD8+ T cells, increased CD4/CD8 ratio, decreased T cell clonality, lower frequencies of top T cell clones in later stages. Further correlation of these immune features with exome sequencing and methylation data demonstrated that the immune response could be impacted by oncogene mutation status, HLA loss, chromosomal copy number aberrations and DNA methylation aberrations suggesting that only cells in preneoplasia with the ideal combination of these molecular features enabling rapid proliferation and evasion from host immune could outgrow into the dominant clones in invasive lung ADCs. Importantly, the immune activation and evasion have started at preneoplastic stage advocating for immunotherapy in patients with lung ADC precursors for prevention of invasive lung cancers.

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“…Every pixel should be classified as a linear combination of spectra using a library as a reference of intensity spectra known to constitute an image (Supplementary Figure S3). In this way, the percentage of each intensity basis spectrum contributing to each pixel can be determined [33]. To generate an appropriate spectral library, uniplex IF staining needs to be performed with each fluorophore used in the panels with a primary antibody (e.g., a cytokeratin, vimentin, CD3, or CD20) without 4 ,6-diamidino-2-phenylindole (DAPI) and with similar dynamic ranges, as described for the uniplex IF staining.…”

Section: Spectral Librarymentioning

confidence: 99%

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies

Parra

Jiang

Solis

et al. 2020

Cancers

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In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

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Multiplex Immunofluorescence Assays

Cited by 54 publications

References 59 publications

Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features

Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features

Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies

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