Background Understanding the spatial distribution of key immune cell populations is critical in advancing our understanding of cancer to inform the development of novel therapeutics. Historically, the spatial analysis of the tumor microenvironment has been limited to relatively low-plex immunohistochemical (IHC) or immunofluorescent (IF) assays, which were inadequate for deep immune cell profiling of the tumor microenvironment.

Methods Here we present the analysis of human breast carcinoma samples with a 60-plex protein assay using a novel precise spatial multiplexing technology called ChipCytometry^TM, which combines iterative immuno-fluorescent staining with high dynamic range imaging to facilitate quantitative phenotyping with single-cell resolution. In addition to traditional pathology review of the high-plex dataset, standard FCS files are generated from multichannel OME-TIFF images, enabling identification and quantification of cellular phenotypes via flow cytometry-like hierarchical gating.

Results The results show precise expression levels for each of the 60 markers in the assay in each individual cell in the sample, maintaining spatial information about each cell. Dozens of immune cell subtypes were identified and quantified based on protein expression profiles. Spatial analysis of the samples reveals quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for in-depth immune profiling in clinical samples.

Conclusions The ChipCytometry platform enables simultaneous detection of multiple protein markers on a single tissue section for deep immune cell profiling in the tumor microenvironment. Combined with the single-cell spatial information, such data sets provide an opportunity for the discovery of new complex multiplexed biomarker signatures to inform therapeutic development.