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MI_intro

 MI_intro

University of Michigan tutorial- Introduction

Julia Wrobel

March 20, 2023
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  1. Julia Wrobel, PhD
    Department of Biostatistics and Informatics
    Introduction to multiplex single-cell
    imaging data

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  2. What is single cell multiplex tissue imaging?
    • High dimensional analysis of tissue samples at the resolution of
    individual cells
    2

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  3. What is single cell multiplex tissue imaging?
    • Single cell refers to individual cell resolution
    3

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  4. What is single cell multiplex tissue imaging?
    • Multiplex refers to multiple types of proteins in the tissue that are
    tagged
    • Each protein label is called a marker
    • Phenotypic markers: used to define cell and/or tissue type
    • Functional markers: inform cell function
    • Present across multiple cell types
    4

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  5. What is single cell multiplex tissue imaging?
    • Multiplex refers to multiple types of proteins in the tissue that are
    tagged
    • Immunofluorescence based
    • Proteins stained with fluorescent antibodies then imaged using
    fluorescence microscopy
    • Mass cytometry based
    • Proteins tagged with metal isotypes (IMC, MIBI)

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  6. What is single cell multiplex tissue imaging?
    • Imaging: biological spatial relationships in tissue are preserved
    • Precursor technologies required suspension of cells in solution,
    destroying spatial info
    6

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  7. What is single cell multiplex tissue imaging?
    • Images produced are multichannel TIFF (.tif) files
    • Each channel is a different protein marker
    • Each pixel contains a continuous intensity value for each marker
    • Example below with non-small cell lung cancer data
    • 8 channels, 3 shown (Left to Right: composite image, nuclei, CK, CD8)
    7

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  8. A brief comparison of MI technologies
    Platform Class Throughput Multiplexicity Software Publications*
    Vectra-Polaris IF high ~8 markers Proprietary > 100
    Discovery
    Ultra
    IF high 5+ markers Limited < 10
    CyTOF
    Imaging
    IMC low 37+ MatLab > 35
    MIBI IMC low 40+ Limited >5
    CODEX Barcode
    -based
    low 40+ Limited/proprietary >1
    * Since April 2020
    8

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  9. Cancer, multiplex imaging, and the tumor
    microenvironment
    The tumor microenvironment (TME) is the
    area within and surrounding a tumor,
    including tumor cells, infiltrating immune
    cells, blood vessels, and other tissue
    • What percentages of immune cell
    subtypes are present before and after
    chemotherapy?
    • Do patients with high spatial clustering of
    B-cells and Macrophages survive longer?
    9

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  10. Multiplex image data processing and analysis pipeline
    Phenotyping
    Segmentation
    Normalization
    Image processing
    Compositional
    data analysis
    Spatial data
    analysis
    Statistical analysis
    Image
    acquisition
    Image
    filtering/
    background
    correction
    Image pre-processing

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  11. Image
    acquisition
    Image
    filtering/
    background
    correction
    Multiplex image data processing and analysis pipeline
    Image
    acquisition
    Image
    filtering/
    background
    correction
    Image pre-processing
    Phenotyping
    Segmentation
    Normalization
    Image processing

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  12. Multiplex image data processing and analysis pipeline
    Cell 1
    Pixel
    1
    Marker
    a
    Marker
    b
    Pixel n
    Marker
    a
    Marker
    b
    Phenotyping
    Segmentation
    Normalization
    Image processing

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  13. Cell types by immune markers
    Thanks to Brooke Fridley and Alex Soupir at the Moffitt Cancer Center for this image

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  14. Multiplex image data processing and analysis pipeline
    Image
    ID
    Cell
    ID
    Tissue type Phenotype X Y Patient
    features
    1 1 Stroma CD4 T-cell … … …
    1 2 Stroma B-cell … … …
    1 3 Tumor Macrophage … … …
    1 4 Stroma CD8 T-cell … … …
    2 1 Tumor Tumor cell … … …
    Phenotyping
    Segmentation
    Normalization
    Image processing

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  15. Multiplex image data processing and analysis pipeline
    Image
    ID
    Cell
    ID
    Tissue type Phenotype X Y Patient
    features
    1 1 Stroma CD4 T-cell … … …
    1 2 Stroma B-cell … … …
    1 3 Tumor Macrophage … … …
    1 4 Stroma CD8 T-cell … … …
    2 1 Tumor Tumor cell … … …
    Compositional
    data analysis
    Spatial data
    analysis
    Statistical analysis

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  16. Multiplex image data processing and analysis pipeline
    Cell type, counts, proportions, or percentages
    Compositional
    data analysis
    Spatial data
    analysis
    Statistical analysis

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  17. Multiplex image data processing and analysis pipeline
    Cell type, counts, proportions, or percentages
    Cell type clustering within an image or across
    patient subgroups
    Compositional
    data analysis
    Spatial data
    analysis
    Statistical analysis

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  18. 1. Quantify characteristics / features of an image
    • Features: cell type proportions, degree of immune cell clustering
    2. Relate to patient-level or clinical outcome
    • Features -> model covariates
    • Outcomes: disease progression, tumor subtype, patient survival time
    How do multiplex images relate to patient outcomes?
    B Steinhart, KR Jordan, J Bapat, MD Post, LW Brubaker, BG Bitler, and J Wrobel. Molecular Cancer Research, 19(12) (2021)

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  19. Shiny Boot Camp:
    Building Interactive Graphics and Dashboards in R
    • July 6-7, 2023; In-person training (NYC)
    • Two-day intensive course including seminars and hands-on coding
    sessions that teaches attendees to build interactive web applications in R
    • Instructor: Julia Wrobel (Colorado School of Public Health)
    • Scholarships available
    More info: publichealth.columbia.edu/Shiny

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