Protocol to quantify immune cell distribution from the vasculature to the glioma microenvironment on sequential immunofluorescence multiplex images
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Updated: Jun 25, 2025
Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment
Published on: March 25, 2020
Hinda Najem1, Sebastian Pacheco1, Joanna Kowal2
1Department of Neurological Surgery, Northwestern University, Chicago, IL 60611, USA; Malnati Brain Tumor Institute of the Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Although myeloid-derived immune cells can be dispersed throughout the tumor microenvironment (TME), anti-tumor effector cells are confined to the perivascular space. Here, we present a protocol to quantify immune cell distribution from tumor vasculature to its glioma microenvironment on sequential immunofluorescence multiplex images. We describe steps for sequential immunofluorescence multiplex staining, image generation, and storage. We then detail the procedures for tissue, vessel, and nuclei segmentation; cell phenotyping; data extraction; and training using RStudio and Spyder.
Although myeloid-derived immune cells can be dispersed throughout the tumor microenvironment (TME), anti-tumor effector cells are confined to the perivascular space. Here, we present a protocol to quantify immune cell distribution from tumor vasculature to its glioma microenvironment on sequential immunofluorescence multiplex images. We describe steps for sequential immunofluorescence multiplex staining, image generation, and storage. We then detail the procedures for tissue, vessel, and nuclei segmentation; cell phenotyping; data extraction; and training using RStudio and Spyder.
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