MARQO pipeline resolves multiparametric cellular and spatial organization in cancer tissue lesions

Buckup, M.; Figueiredo, I.; Ioannou, G.; Ozbey, S.; Cabal, R.; Tabachnikova, A.; Troncoso, L.; Le Berichel, J.; Zhao, Z.; Ward, S. C.; Hennequin, C.; Akturk, G.; Hamel, S.; Fiel, M. I.; Brody, R.; Schwartz, M.; Marron, T. U.; Kim-Schulze, S.; Gonzalez-Kozlova, E.; Roudko, V.; Hamon, P.; Merad, M.; Gnjatic, S.

doi:10.1101/2025.04.24.650539

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bioRxiv · 10.1101/2025.04.24.650539

MARQO pipeline resolves multiparametric cellular and spatial organization in cancer tissue lesions

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Abstract

Multiplex immunostaining analysis remains fragmented, underperforming, and labor-intensive despite tissue proteomic methodologies achieving ever-increasing marker complexity. Here we propose an open-source, semi-supervised automated pipeline that streamlines start-to-finish, single-cell resolution analysis of whole-slide tissue, named Multiplex-imaging Analysis, Registration, Quantification, and Overlaying (MARQO). We compared and validated MARQO using Multiplex Immunohistochemical Consecutive Staining on a Single Slide (MICSSS) using human tumor and adjacent normal tissue samples. Performance was compared with manually-curated pathologist determinations and quantification of multiple markers. We also optimized MARQO to analyze diverse tissue sizes (whole tissue, biopsy, tissue microarray) and staining approaches (singleplex immunohistochemistry, 20-color multiplex immunofluorescence) to determine marker co-expression patterns in multiple human solid cancer types. Lastly, we validated CD8 T cell enrichment in hepatocellular carcinoma responders to neoadjuvant cemiplimab in a phase II clinical trial, further demonstrating MARQOs ability to provide spatially-resolved in situ mechanisms by providing multiplex whole-slide single-cell resolution data.

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Buckup, M., Figueiredo, I., Ioannou, G., Ozbey, S., Cabal, R., Tabachnikova, A., Troncoso, L., Le Berichel, J., Zhao, Z., Ward, S. C., Hennequin, C., Akturk, G., Hamel, S., Fiel, M. I., Brody, R., Schwartz, M., Marron, T. U., Kim-Schulze, S., Gonzalez-Kozlova, E., Roudko, V., Hamon, P., Merad, M., Gnjatic, S.. 2025-04-26. MARQO pipeline resolves multiparametric cellular and spatial organization in cancer tissue lesions. https://doi.org/10.1101/2025.04.24.650539

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