The InSituPlex® Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples

Manesse, Maël; Patel, Katir; Bobrow, Mark; Downing, Sean R.

doi:10.1007/978-1-4939-9773-2_26

Cited by 43 publications

(34 citation statements)

References 20 publications

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“…Four antibodies are detected simultaneously with fluorophore‐labeled complementary oligonucleotide (probe) strands that hybridize with their target oligonucleotides' DNAs. After nuclear counterstaining, a coverslip is applied and the tissue is imaged (see Figure 1D) [5]. To achieve higher order (>4 antibodies) multiplexing, the coverslip is removed; the sample is de‐hybridized to remove the existing fluorescent probe strands; and the next set of four probe strands are hybridized to detect the second set of markers that was incubated and amplified in the first staining reaction.…”

Section: Ultivuementioning

confidence: 99%

Clinical and research applications of multiplexed immunohistochemistry and in situ hybridization

McGinnis¹,

Ibarra-Lopez²,

Rost³

et al. 2021

The Journal of Pathology

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Over the past decade, invention and adoption of novel multiplexing technologies for tissues have made increasing impacts in basic and translational research and, to a lesser degree, clinical medicine. Platforms capable of highly multiplexed immunohistochemistry or in situ RNA measurements promise evaluation of protein or RNA targets at levels of plex and sensitivity logs above traditional methods – all with preservation of spatial context. These methods promise objective biomarker quantification, markedly increased sensitivity, and single‐cell resolution. Increasingly, development of novel technologies is enabling multi‐omic interrogations with spatial correlation of RNA and protein expression profiles in the same sample. Such sophisticated methods will provide unprecedented insights into tissue biology, biomarker science, and, ultimately, patient health. However, this sophistication comes at significant cost, requiring extensive time, practical knowledge, and resources to implement. This review will describe the technical features, advantages, and limitations of currently available multiplexed immunohistochemistry and spatial transcriptomic platforms. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Section: Ultivuementioning

confidence: 99%

Clinical and research applications of multiplexed immunohistochemistry and in situ hybridization

McGinnis¹,

Ibarra-Lopez²,

Rost³

et al. 2021

The Journal of Pathology

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“…Such techniques identify biomarker co-expression on individual cells, reveal the spatial distribution of immune, malignant, and stromal cells and reduce consumption of precious tissue specimens. Beyond employing conventional chromogenic detection systems or fluorescently labelled antibodies, newer techniques that assess multiple biomarkers in situ take advantage of DNA barcoded [84,85] or metal-tagged (Imaging Mass Cytometry [86] and Multiplexed Ion Beam Imaging [87]) antibodies [88]. New studies using multiplex approaches are identifying specific immune cells in the tumor microenvironment of sarcomas with predictive and prognostic value [33,47,89].…”

Section: New Technologiesmentioning

confidence: 99%

Sarcomas: Immune biomarker expression and checkpoint inhibitor trials

Zhu

Shenasa

Nielsen

2020

Cancer Treatment Reviews

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Sarcomas are a heterogenous group of mesenchymal cancers comprising over 100 subtypes. Current chemotherapy for all but a very few subtypes has limited efficacy, resulting in 5-year relative survival rates of 16% for metastatic patients. While sarcomas have often been regarded as an "immune cold" tumor category, recent biomarker studies have confirmed a great deal of immune heterogeneity across sarcoma subtypes. Reports from the first generation of clinical trials treating sarcomas with immunotherapy demonstrate a few positive responses, supporting efforts to stratify patients to optimize response rates. This review summarizes recent advances in knowledge around immune biomarker expression in sarcomas, the potential use of new technologies to complement these study results, and clinical trials particularly of immune checkpoint inhibitor therapy in sarcomas.Each of the immune biomarkers assessed was reviewed for subtype-specific expression patterns and correlation with prognosis. Overall, there is extensive heterogeneity of immune biomarker presence across sarcoma subtypes, and no consensus on the prognostic effect of these biomarkers. New technologies such as multiplex immunohistochemistry and high plex in situ profiling may offer more insights into the sarcoma microenvironment. To date, clinical trials using immune checkpoint inhibitor monotherapy have not shown compelling clinical benefits. Combination therapy with dual checkpoint inhibitors or in combinations with other agents has yielded more promising results in dedifferentiated liposarcoma, undifferentiated pleomorphic sarcoma, angiosarcoma and alveolar soft-part sarcoma. Better understanding of the sarcoma immune status through biomarkers may help decipher the reasons behind differential responses to immunotherapy.

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“…Direct immunofluorescence using fluorophore-conjugated primary antibodies and chemical inactivation of fluorescent dyes enables detection of over 50 protein targets in a single tissue section, in cyclic immunofluorescence (CyCIF) [7][8][9] and NeoGenomics' MultiOmyx platform 10 . Furthermore, fluorophore-conjugated DNA barcodes (i.e., oligonucleotides) facilitate multiplexing in co-detection by indexing (Akoya's CODEX) 11 , Ultivue's InSituPlex 12 , and Ab-oligo cyCIF 13 . Imaging mass cytometry 14 and multiplex ion beam imaging 15 can Adopting the method of Lin et al 7 , we stained formalin-fixed, paraffin-embedded (FFPE) tissues using a cyclic process in which four proteins per cycle were labeled using a direct immunofluorescence (IF) protocol, imaged, and quenched of fluorescence signal (Fig.…”

Section: Introductionmentioning

confidence: 99%

A framework for multiplex imaging optimization and reproducible analysis

Eng

Bucher

et al. 2021

Preprint

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Multiplex imaging technologies are increasingly used for single-cell phenotyping and spatial characterization of tissues; however, transparent methods are needed for comparing the performance of platforms, protocols and analytical pipelines. We developed a python software, jinxif, for reproducible image processing and utilize Jupyter notebooks to share our optimization of signal removal, antibody specificity, background correction and batch normalization of the multiplex imaging with a focus on cyclic immunofluorescence (CyCIF). Our work both improves the CyCIF methodology and provides a framework for multiplexed image analytics that can be easily shared and reproduced.

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The InSituPlex® Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples

Cited by 43 publications

References 20 publications

Clinical and research applications of multiplexed immunohistochemistry and in situ hybridization

Clinical and research applications of multiplexed immunohistochemistry and in situ hybridization

Sarcomas: Immune biomarker expression and checkpoint inhibitor trials

A framework for multiplex imaging optimization and reproducible analysis

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