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Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens
Published on: January 21, 2019
Highly Multiplexed Immunofluorescence PhenoCycler Panel for Murine Formalin-Fixed Paraffin-Embedded Tissues Yields
Sachin S Surwase1, Xin Ming M Zhou2, Kathryn M Luly1
1Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Insights
We developed a new spatial proteomics panel for formalin-fixed tissues, enabling detailed analysis of the tumor immune microenvironment. This panel revealed how gene delivery nanoparticles and anti-PD1 therapy impact melanoma, offering insights into treatment efficacy.
Area of Science:
- Spatial proteomics and multiplex immunofluorescence imaging.
- Tumor microenvironment analysis in cancer research.
- Preclinical research applications of advanced imaging techniques.
Background:
- Spatial proteomics profiling reveals cell types and interactions within tissue microenvironments.
- PhenoCycler is a multiplex immunofluorescence imaging technology using oligonucleotide-barcoded antibodies.
- Existing PhenoCycler panels are mainly for fresh frozen tissues, limiting use with formalin-fixed paraffin-embedded (FFPE) tissues.
Purpose of the Study:
- To present a novel PhenoCycler panel of 28 validated antibodies for murine FFPE tissues.
- To describe the workflow for antibody validation, barcoding, panel design, and multiplex imaging.
- To apply the panel and analysis pipeline to investigate nanoparticle formulations in melanoma immunotherapy.
Main Methods:
- Selection and validation of antibody clones for FFPE tissues.
- Oligonucleotide-barcoding of antibodies for cyclic immunofluorescence.
- Multiplex imaging using the PhenoCycler system.
- Single-cell proteomics data analysis, including clustering and spatial relationship quantification.
Main Results:
- Gene delivery of 4-1BBL and IL-12 shifted macrophage polarization and increased CD8 T cell-macrophage interactions.
- Adding interferon gamma enhanced antigen presentation and PD-L1 expression.
- Interferon gamma also promoted closer CD8 T cell and tumor cell interactions, potentially explaining therapeutic limitations.
Conclusions:
- The developed PhenoCycler panel is effective for analyzing murine FFPE tissues.
- Gene delivery strategies can modulate the tumor immune microenvironment.
- Understanding these spatial dynamics provides mechanistic insights into immunotherapy efficacy and limitations.
Abstract:
Spatial proteomics profiling is an emerging set of technologies that has the potential to elucidate the cell types, interactions, and molecular signatures that make up complex tissue microenvironments, with applications in the study of cancer, immunity, and much more. An emerging technique in the field is CoDetection by indEXing, recently renamed as the PhenoCycler system. This is a highly multiplexed immunofluorescence imaging technology that relies on oligonucleotide-barcoded antibodies and cyclic immunofluorescence to visualize many antibody markers in a single specimen while preserving tissue architecture. Existing PhenoCycler panels are primarily designed for fresh frozen tissues. Formalin-fixed paraffin-embedded blocks offer several advantages in preclinical research, but few antibody clones have been identified in this setting for PhenoCycler imaging. Here, we present a novel PhenoCycler panel of 28 validated antibodies for murine formalin-fixed paraffin-embedded tissues. We describe our workflow for selecting and validating clones, barcoding antibodies, designing our panel, and performing multiplex imaging. We further detail our analysis pipeline for comparing marker expressions, clustering and phenotyping single-cell proteomics data, and quantifying spatial relationships. We then apply our panel and analysis protocol to profile the effects of 3 gene delivery nanoparticle formulations, in combination with systemic anti-PD1, on the murine melanoma tumor immune microenvironment. Intralesional delivery of genes expressing the costimulatory molecule 4-1BBL and the cytokine IL-12 led to a shift toward intratumoral M1 macrophage polarization and promoted closer associations between intratumoral CD8 T cells and macrophages. Delivery of interferon gamma, in addition to 4-1BBL and IL-12, not only further increased markers of antigen presentation on tumor cells and intratumoral antigen-presenting cells but also promoted greater expression of checkpoint marker PD-L1 and closer associations between intratumoral CD8 T cells and PD-L1-expressing tumor cells. These findings help explain the benefits of 4-1BBL and IL-12 delivery while offering additional mechanistic insights into the limitations of interferon gamma therapeutic efficacy.
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