Ly, C.; Veletic, I.; Pacheco, C.; Dasdemir, E.; Jelloul, F. Z.; Ferri-Borgogno, S.; Basi, A. V.; Gomez, J. A.; Root, J. L.; Reville, P. K.; Sharma, P.; Basu, S.; Quesada, A. E.; Bueso-Ramos, C.; Manshouri, T.; Garcia, M. B.; Burks, J. K.; Abbas, H.

Acute myeloid leukemia (AML) resides in an immune rich microenvironment, yet, immune-based therapies have faltered in eliciting durable responses. Bridging this paradox requires a comprehensive understanding of leukemic interactions within the bone marrow microenvironment. We optimized a high-throughput tissue-microarray based pipeline for high-plex spatial immunofluorescence and mass cytometry imaging on a single slide, capturing immune, tumor, and structural components. Using unbiased clustering on the spatial K function, we unveiled the presence of tertiary lymphoid-like aggregates in bone marrow which we validated using spatial transcriptomics and an independent proteomics approach. We then found validated TLS signatures predictive of outcomes in AML using an integrated public 480 patient transcriptomic dataset. By harnessing high-plex spatial proteomics, we open the possibility of discovering of novel structures and interactions that underpin leukemic immune response. Further, our study\'s methodologies and resources can be adapted for other bone marrow diseases where decalcification and autofluorescence present challenges.