Integration of spatial single-cell proteomics and spatial metabolomics reveals tumor microenvironment predictive of immunotherapy response in mucosal melanoma

Wang, J.; Nagarajan, P.; Cho, S.; Liu, Y.; Seeley, E. H.; Dai, Y.; Yu, K.; Burks, J. K.; McQuade, J. L.; Diab, A.; Wang, L.; Ekmekcioglu, S.

doi:10.64898/2026.04.23.720453

bioRxiv · 10.64898/2026.04.23.720453

Integration of spatial single-cell proteomics and spatial metabolomics reveals tumor microenvironment predictive of immunotherapy response in mucosal melanoma

Wang, J.·Nagarajan, P.·Cho, S.·Liu, Y.·Seeley, E. H.·Dai, Y.·Yu, K.·Burks, J. K.·McQuade, J. L.·Diab, A.·Wang, L.·Ekmekcioglu, S.

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Abstract

Mucosal melanoma (MuM) is a rare but aggressive malignancy with limited benefit from immune checkpoint inhibition and few predictive biomarkers. We integrated single-cell spatial proteomics (COMET) and spatial metabolomics (MALDI-IMS) to profile 97 tissue cores from 26 patients treated with PD-1/PD-L1 and/or CTLA-4 inhibitors. We profiled 695,444 cells and resolved 25 cell states across eight major cell types. Cellular neighborhood (CN) analysis revealed distinct tumor- and stromal-associated spatial architectures. Responders were enriched for tumor-associated CNs (invasive tumor and tumor boundary) with close spatial proximity among Ki67 tumor cells, CD163 macrophages, and CD11c dendritic cells (DCs), and increased proliferating/cytotoxic CD8+ T-cell subsets. Non-responders showed stromal CN dominance with reduced immune infiltration. Spatial metabolomics identified lower abundance of indole-derived metabolites and reduced indole/tryptophan pathway activity in responders within tumor and TME regions that tracked with DC/macrophage-enriched spatial contexts. This study advances MuM spatial biology and provides a framework for biomarker-driven immunotherapy strategies. Statement of significanceMucosal melanoma (MuM) responds poorly to immune checkpoint blockade, and predictive biomarkers are limited. Integrated spatial proteomics and metabolomics reveal response-associated tumor-immune neighborhood architecture, stromal contexts linked to immune exclusion, and altered indole/tryptophan metabolism in the microenvironment. These spatial features nominate biomarkers and therapeutic hypotheses to improve immunotherapy for MuM.

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Wang, J., Nagarajan, P., Cho, S., Liu, Y., Seeley, E. H., Dai, Y., Yu, K., Burks, J. K., McQuade, J. L., Diab, A., Wang, L., Ekmekcioglu, S.. 2026-04-27. Integration of spatial single-cell proteomics and spatial metabolomics reveals tumor microenvironment predictive of immunotherapy response in mucosal melanoma. https://doi.org/10.64898/2026.04.23.720453

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