Highly multiplexed spatial biomarker analysis has demonstrated the potential to advance our current understanding of the immune system and its role in cancer – from tumor initiation to metastatic progression. Previously, a trade-off between plex and spatial context meant that our understanding of immune cell involvement in cancer was limited by either single-plex technologies with spatial context (i.e., immunohistochemistry) or highly multiplex technologies without spatial context (i.e., single-cell RNA sequencing). ChipCytometryTM is a novel, highly multiplexed technology that preserves both plex and spatial context to deeply profile immune cell diversity at single-cell resolution. ChipCytometry uses commercially available antibodies and combines iterative immuno-fluorescent staining with high-dynamic range imaging to profile dozens of protein biomarkers in a single tissue specimen. Cellular phenotypes are identified with via flow cytometry-like hierarchical gating from standard multichannel OME-TIFF images, compatible with a variety of computational tools being developed for multiplexed analysis and visualization. Here, we use ChipCytometry to identify and quantify key immune cell subtypes in both normal (i.e., appendix) and tumor tissues (i.e., melanoma and non-small cell lung cancer) FFPE samples. The results show precise expression levels for each biomarker in the assay in each individual cell in the sample, while maintaining spatial positioning of each cell. Spatial analysis reveals quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for the in-depth immune profiling in clinical FFPE samples.

Cancer therapies that rely on manipulating or engineering immune cells have shown much promise in recent years for effectively combating a broad array of cancer types. In order to determine the effectiveness of such therapies, it is necessary to accurately profile the complement of immune cell types present in the tumor microenvironment. Methods to reliably obtain immune cell signatures require the combination of powerful resolution to discriminate cell boundaries, broad dynamic range to capture markers of varying expression levels, and accurate cell segmentation across a wide range of cell sizes and morphologies. Many methods for quantitative immune cell profiling have demonstrated shortcomings when it comes to the aforementioned attributes. To remedy these shortcomings, we present here ChipCytometry, an imaging technology for immune profiling of cells and sectioned tissues. ChipCytometry is a fluorescence-based imaging system that utilizes multiplexed immuno-fluorescence staining in combination with high-dynamic-range (HDR) imaging to facilitate quantitative phenotyping of individual cells within tissue samples. Employing this technology to profile metastatic and primary tumors, multiple biomarkers were stained in iterative multiplex assays to profile single cells with an AI-powered cell segmentation algorithm. The HDR imaging allows for both strong and weak fluorescent signals to be simultaneously obtained without loss of sensitivity. This immune profiling was completed on 6 different cryosectioned human tumor tissues (head & neck, liver, lung, breast, colon, and pancreas).

Immunohistochemistry is the most widely used diagnostic technique in tissue pathology. However, IHC is associated with several limitations including the labeling of just a few markers per tissue section and limited quantification of cell populations. As a result of plex limitations, key insights about tumor biology are missed, which could be important for advancing our understanding of tumor biology and ultimately improving patient outcomes. Chip Cytometry is a novel image-based platform for precise spatial multiplexing that addresses these challenges by combining iterative immuno-fluorescent staining with high-dynamic range imaging to facilitate quantitative phenotyping with single-cell resolution. The platform enables simultaneous detection of dozens of markers on a single tissue section and enables accurate quantification of protein expression levels necessary to deeply profile single cells, understand interactions between key immune cells, and identify topographic biomarkers. Here we demonstrate how standard FCS files are generated from multichannel OME TIFF images, enabling identification of cellular phenotypes via flow cytometry-like hierarchical gating. Quantification of results reveal precise expression levels for each marker in the assay in each individual cell in the sample, while maintaining spatial information about each cell. Chip Cytometry has the potential to advance precision medicine in immuno-oncology and inform the discovery of novel biomarkers by enabling quantitative analysis of cellular phenotypes in the spatial context. The Chip Cytometry 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. Citation Format: Karen Kwarta, Thomas Campbell, Adam Northcutt, Spencer Schwarz. Precise spatial multiplexing of protein biomarkers for immune profiling in tissue samples with chip cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6769.