2.3. Antibody Conjugated with DNA-Barcode

CODEX^® (Akoya Biosciences, Marlborough, MA, USA) is a commercially available method that uses DNA conjugated antibodies [41,42]. CODEX detects antibodies labeled with unique DNA-barcodes. The detection is highly specific and can simultaneously capture the expression of more than 40 biomarkers. Tissue FFPE slides are stained in a single step with the antibody panel. Consecutive cycles of labeling, imaging and removing barcodes are performed in a fully automatic way by the CODEX fluidics instrument. The collected images are then integrated into one, and image analysis is performed by CODEX analysis manager and CODEX Analysis Viewer software. Tissue samples remain undisrupted and can be reused for future studies. This technique provides the opportunity for simultaneous assessment of expression and spatial distribution of multiple biomarkers in situ on a single slide. CODEX can achieve spatial resolution at a level as low as 0.26 μm, making single cell resolution possible. It has the advantage of being compatible with existing fluorescent microscopes for image generation. Although this platform can achieve comprehensive high-level multiplexing, its use is held back by its high cost, limited throughput and restriction to only small region of interest (ROI) on the slide available for the imaging. InSituPlex^® (Ultivue, Cambridge, MA, USA) is another multiplex in situ method that uses antibodies conjugated with DNA-barcodes [43]. Tissue FFPE slides are incubated with the primary antibody mixture in one step. After barcode amplification, a mixture of complementary, fluorophore-linked probes hybridizes each barcode, augmenting the signal. The technique is compatible with various scanning and image analysis systems.

GeoMx^® Digital Spatial Profiling (DSP) (Nanostring, Seattle, WA, USA) is a commercially available method that uses DNA-barcode conjugated antibodies [44,45]. In this platform, two types of antibodies are used: compartment-defining antibodies conjugated with fluorophores to morphologically select ROIs, and antibodies conjugated with unique oligonucleotide barcodes for multiplexing. This high-throughput technique provides simultaneous assessment of expression of multiple biomarkers in situ and in a compartment-based manner within selected ROIs, determined by fluorescently conjugated antibodies. Tissue slides are stained in a single step with the antibody mastermix. Then, UV light cleaves the barcodes from the antibodies, and the barcodes are collected in a microplate and quantified by the commercially available NanoString nCounter system. After quantitative analysis, the pool of antibodies originate back to their corresponding region of interest to allow spatial characterization. The detection can capture the expression of more than seventy biomarkers at the protein level, despite considerable amount of non-specific antibody binding and limited number of validated antibodies, although the various unique Nanostring barcodes. DSP could be useful for evaluating the expression of multiple immune regulators, since the spatial heterogeneity is addressed by examining stromal and tumoral compartments, as well as multiple ROIs. However, DSP provides limited spatial characterization of cell immunophenotypes, due to narrow “morphology-panel” selection, consisting mainly by three or four markers, such as cytokeratin and CD45 [38]. Hence, within a ROI, the DSP method could potentially suffer from missing rare cellular subpopulations, as in bulk-omics methods. Heatmaps of biomarker expression can be used to “visualize” the ROI but such an approach is costly for a whole slide. Although this platform can achieve comprehensive high-level multiplexing, high cost and limited spatial information represent some of its drawbacks.