The bone marrow (BM) microenvironment consists of various cell types such as mesenchymal stromal cells, endothelial cells, osteoblastic cells and multiple immune cell types including mature myeloid cells and lymphocytes. Recent studies have shown that leukemias can create and maintain a leukemia-supporting BM microenvironment, and vice versa, a dysfunctional BM microenvironment can contribute to leukemia development and progression. Moreover, in tumors the microenvironment is often immunosuppressive and restrains effective anti-tumoral immune responses by adaptive and innate immunity. A better understanding of the precise localization of microenvironmental and immune cell types in intact tissue, and how they physically interact with each other and with tumor cells, will improve our understanding of the mechanisms by which cancer reprograms its microenvironment and may form the basis for novel immunotherapies.


CO-Detection by antibody indEXing (CODEX) is a multiplex fluorescence microscopy platform based on DNA-conjugated antibodies that allows the analysis of 50+ markers in a single tissue section. After staining with an antibody cocktail, tissues are imaged in a multi-cycle reaction using a microfluidics system and a fluorescence microscope with a computer automated X/Y/Z stage. DNA-conjugated antibodies are rendered visible using complementary fluorescent DNA probes, followed by imaging, probe stripping, washing and re-rendering. This process is repeated until all the antibodies present in the initial cocktail have been rendered and imaged. Here, we used CODEX to analyze intact BM at the single-cell level (~200nm resolution) during leukemic progression. Chronic myeloid leukemia (CML)-like disease was induced in non-irradiated mice using BCR-ABL1-GFP retrovirus. Tissue sections of femoral bones harvested at different time points after leukemia onset were stained using a 50+ marker CODEX antibody panel to simultaneously identify hematopoietic and leukemic stem and progenitor cells, multiple BM microenvironmental cell types, myeloid and lymphoid cells as well as functional markers.


We have built an integrated computational pipeline for the analysis of high-dimensional CODEX data that enables the identification and characterization of BM cell types as well as their spatial organization in situ. Raw images were concatenated and aligned using Hoechst nuclear stain as a reference, followed by deconvolution, segmentation, marker expression quantification and spatial compensation. Exported data were clustered in an unsupervised manner using VorteX algorithm, which identified 28 distinct cellular clusters based on marker expression values. All major BM compartments including stromal (vascular, pericytes, osteoblastic), lymphoid (T and B cell subsets), myeloid (megakaryocytes, macrophages, dendritic cells, granulocytes) and progenitor cell types, as well as leukemic cells, were represented. During leukemic progression, the BM microenvironment was dramatically rearranged. Besides the expected growth of the leukemic clone, we observed a massive increase in vascular and osteoblastic cell types, whereas immune cell clusters were significantly reduced. Interestingly, CD71, the transferrin receptor, was strongly up-regulated on tumor cells in advanced leukemia, indicating towards a role for iron metabolism in malignant progression. Furthermore, hierarchical clustering of tissue regions based on cellular composition using X/Y/Z positional information pointed towards the emergence of specific cell-cell interaction modules that developed during leukemic progression, including mutual attraction between B cells and central arterioles.


High-dimensional imaging of the BM microenvironment by CODEX allows studying the abundance and distribution of cellular elements that are often underestimated or missed by traditional flow cytometry, such as stromal cells, vasculature and megakaryocytes. Importantly, CODEX identifies single cells in their tissue context during leukemic progression and facilitates the discovery of novel cell-cell interactions and cell types as well as unexpected marker constellations.


Samusik:Akoya Biosciences: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Nolan:Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Goltsev:Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Author notes


Asterisk with author names denotes non-ASH members.

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