Spatial Phenotyping Adds a New Dimension to Discovery Biology

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It is well known that cells don’t live alone. Every cell exists within a dynamic, three-dimensional microenvironment. The interplay between neighboring cells or groups of cells can have a tremendous impact on important biological processes. Will a CD4 T-cell in breast cancer tissue surrounded by CD8 T cells behave differently than a CD4 T-cell surrounded by a bunch of luminal epithelial cells? Same cell, different context—that’s the idea behind spatial biology and spatial phenotyping.

Why context matters

Knowing how different types of cells are organized in space provides powerful information about the microenvironment. Consider tumor immunotherapy. For a decade, immune checkpoint inhibitors have shown remarkable antitumor effects, but only in a minority of patients. Researchers are on the hunt for biomarkers that will signal which patient will likely benefit from immune checkpoint blockade.

Looking at biomarkers averaged across the entire sample can’t provide that answer. Two samples may contain the same types of immune cells, but the relative positions of those immune cells can reveal a great deal about the tumor prognosis. Where are the immune cells relative to the tumor cells? Where are the immunosuppressive cells? Sequencing alone can’t provide that information. Multiplexed imaging technologies allow researchers to visualize dozens of biomarkers at a time while preserving their context.


Spatial phenotyping via multiplex imaging tools like CODEX® provides the advantages of single-cell analysis while preserving spatial relationships between the cells. This offers an invaluable tool for uncovering novel insights in research areas such as neuroscience, immunology, and cancer research.

Akoya Biosciences commercialized the CODEX solution in early 2019. Initially developed in the lab of Garry Nolan, PhD, at Stanford University, CODEX enables imaging of more than 40 biomarkers at a time, using a panel of customizable antibodies. Each antibody is tagged with a unique oligonucleotide, and the antibodies are visualized using complementary oligos conjugated to fluorophores.

Three spectrally distinct dyes can be applied at one time. These reporters are hybridized to the antibodies, imaged, and then gently removed so another set of reporters can be applied. The CODEX instrument cycles through this process of hybridizing, imaging, and removing until all the antibodies have been imaged.

Cellular neighborhoods—the new spatial phenotype

Recently, the Nolan lab used CODEX to study colorectal cancer. In their analysis of the multiplexed imaging data, the researchers discovered “cellular neighborhoods,” collections of components characteristic of the cancer immune tumor environment.

voronoi diagrams
Representative voronoi diagrams of the nine different cellular neighborhoods (left) and corresponding seven-color CODEX® image (right) discovered by the Nolan lab. Schurch CM et al. Cell 2020;182(5):1341-1359.

The team defined nine distinct cellular neighborhoods and observed the interactions between them. Neighborhoods were based on the presence of different types of immune cells and their typical environments. They compared these cellular neighborhoods across two types of colorectal cancers, i.e., a Crohn’s disease-like reaction, which has good survival, and diffuse immune infiltrate, which has poor outcomes.

Nolan’s work showed that the distance between immune cells can be highly predictive. For instance, the closer the effector cells were to the tumor cells relative to their distance from the immunosuppressive (Treg) cells, the more likely it was that anti-PD-1 therapy would succeed. These metrics were only discoverable with spatial biology.

The tumor microenvironment is one notable example where spatial biology uncovers new discoveries in tissues.  We are just beginning to understand the applications of spatial biology and its potential provide a new paradigm for tissue discoveries.


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