Multiplex Immunofluorescence and Multispectral Imaging: Forming the Basis of a Clinical Test Platform for Immuno-Oncology

Abstract

As immuno-oncology (I/O) emerges as an effective approach in the fight against cancer, multispectral imaging of multiplex immunofluorescence (mIF) is maturing as an analytical platform. The timing is fortuitous. Due to health economic considerations surrounding the use of I/O, there is an urgent need for tests that accurately predict response to the growing list of available therapies. Multispectral mIF provides several advantages over other biomarker modalities by enabling deeper interrogation of the intricate biology within the tumor microenvironment, including detection of cell-to-cell spatial interactions that correlate with clinical outcomes. It also provides a practical path for generating reliable and reproducible results in a clinically suitable, high-throughput workflow. In this article, we (1) describe the principles behind multispectral mIF; (2) provide advice and recommendations on assay development and optimization and highlight characteristics of a well-performing assay; and (3) discuss the requirements for translating this approach into clinical practice.

Importance of the Immune Microenvironment in the Spontaneous Regression of Cervical Squamous Intraepithelial Lesions (cSIL) and Implications for Immunotherapy

“…mIF is an efficient and reliable technique to monitor the immune microenvironment. In addition to complex cell-to-cell biology, spatial interactions can be studied in great detail [ 135 , 162 ]. Next to immune biomarkers, other possible viral and host factors should be further explored investigating the interaction with the lesion microenvironment and the way the immune system responds to HPV.…”

Combined protein and nucleic acid imaging reveals virus-dependent B cell and macrophage immunosuppression of tissue microenvironments

“…Here, we present an approach, protein and nucleic acid in situ imaging (PANINI), that consists of a (1) highly sensitive custom branched-chain amplification method for nucleic acid targets using a tyramide-based amplification to deposit haptens, which are then detected using antibodies conjugated to lanthanide or oligo tags for a further amplification step, (2) an optimized antigen retrieval protocol that bypasses the protease treatment and yet allows nucleic acid detection down to a single genomic event, and (3) an antibody-based detection of nucleic acid targets and proteins by using the MIBI ( Keren et al., 2019 ), Vectra Polaris ( Hoyt, 2021 ), and CODEX platforms ( Goltsev et al., 2018 ). Using FFPE cell pellets and lymphoid tissues from simian immunodeficiency virus (SIV)-infected and SIV-uninfected rhesus macaques, we demonstrate that PANINI-MIBI is capable of simultaneous detection of single-integration events of SIV DNA (vDNA), SIV RNA transcripts (vRNA), and protein epitopes robustly on the same tissue section.…”

Navigating the cellular landscape in tissue: Recent advances in defining the pathogenesis of human disease

“…A robust and objective workflow for imaging data analysis and visualization still needs to be established, especially if these methods are developed for clinical use [18] , [80] , [81] . Robust and standardized workflows from panel design and imaging to data acquisition are fundamental in ensuring these results are valid and are reproducible across different tissue samples and studies [7] , [22] . As summarized in Fig.…”

“…This allows the detection of markers with low expression and the use of primary antibodies from the same species, while still maintaining a high signal-to-noise ratio [16] , [17] , [19] , [20] , [21] . TSA is also more photostable compared to conventional immunofluorescence, which enables the long-term storage and re-imaging of the slides after the initial staining [22] .

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