Fig. 1: MARQO pipeline overview.

Variable formalin-fixed and paraffin-embedded (FFPE) tissue sizes from small TMA cores to whole-slide tissue resections can be analysed using a tiling architecture, which can be performed locally or externally via a cluster. The pipeline is compatible for slides stained with MICSSS, singleplex IHC, or mIF and CyCIF. After the user provides a brief quality control when importing data, the pipeline independently processes each staining technology differently. MICSSS and IHC undergo dynamic deconvolution to extract nuclear and chromogen staining. All technologies are converted into numerous smaller, overlapping tiles for faster, parallel processing. The MICSSS and CyCIF technologies undergo a series of registrations. All technologies are segmented for cell nuclei, where MICSSS uses a composite methodology from its iterative nuclear staining. All technologies quantify the chromogenic positivity staining, nuclear staining and morphological information. These metadata are used for cluster-based classification, after which the user quality controls to assess which markers the clusters truly stain positively or negatively for. The user can use MARQO downstream analysis tools to further review data. These modules can reconcile tissue compartment annotations from QuPath, recreate large chromogenic or whole-channel image files that combine multiple individual tiles, create figure-ready qualitative overlays to visualize cell populations and export data for third-party programs. At multiple checkpoints in the pipeline, intermediary files that are also easily integrated into third-party programs are exported. Illustrations created with BioRender.com.