While the bone marrow attracts tumor cells in many solid cancers leading to poor outcome in affected patients, comprehensive analyses of bone marrow metastases have not been performed on a single-cell level. We here set out to capture tumor heterogeneity and unravel microenvironmental changes in neuroblastoma, a solid cancer with bone marrow involvement. To this end, we employed a multi-omics data mining approach to define a multiplex imaging panel and developed DeepFLEX, a pipeline for subsequent multiplex image analysis, whereby we constructed a single-cell atlas of over 35,000 disseminated tumor cells (DTCs) and cells of their microenvironment in the metastatic bone marrow niche. Further, we independently profiled the transcriptome of a cohort of 38 patients with and without bone marrow metastasis. Our results revealed vast diversity among DTCs and suggest that FAIM2 can act as a complementary marker to capture DTC heterogeneity. Importantly, we demonstrate that malignant bone marrow infiltration is associated with an inflammatory response and at the same time the presence of immuno-suppressive cell types, most prominently an immature neutrophil/granulocytic myeloid-derived suppressor-like cell type. The presented findings indicate that metastatic tumor cells shape the bone marrow microenvironment, warranting deeper investigations of spatio-temporal dynamics at the single-cell level and their clinical relevance.

Bone marrow commonly serves as a metastatic niche for disseminated tumor cells (DTCs) of solid cancers in patients with unfavorable clinical outcome. Single-cell assessment of bone marrow metastases is essential to decipher the entire spectrum of tumor heterogeneity in these cancers, however, has previously not been performed. Here we used multi-epitope-ligand cartography (MELC) to spatially profile 20 biomarkers and assess morphology in DTCs as well as hematopoietic and mesenchymal cells of eight bone marrow metastases from neuroblastoma patients. We developed DeepFLEX, a single-cell image analysis pipeline for MELC data that combines deep learning-based cell and nucleus segmentation and overcomes frequent challenges of multiplex imaging methods including autofluorescence and unspecific antibody binding. Using DeepFLEX, we built a single-cell atlas of bone marrow metastases comprising more than 35,000 single cells. Comparisons of cell type proportions between samples indicated that microenvironmental changes in the metastatic bone marrow are associated with tumor cell infiltration and therapy response. Hierarchical clustering of DTCs revealed multiple phenotypes with highly diverse expression of markers such as FAIM2, an inhibitory protein in the Fas apoptotic pathway, which we propose as a complementary marker to capture DTC heterogeneity in neuroblastoma. The presented single-cell atlas provides first insights into the heterogeneity of human bone marrow metastases and is an important step towards a deeper understanding of DTCs and their interactions with the bone marrow niche.

Neutrophils are evolutionarily conserved innate defense cells implicated in diverse pathological processes. Zebrafish models have contributed substantially to our understanding of neutrophil functions, but similarities to human neutrophil maturation have not been characterized limiting applicability to study human disease. We generated transgenic zebrafish strains to distinguish neutrophil maturation grades in vivo and established a high-resolution transcriptional profile of neutrophil maturation. We linked gene expression at each stage to characteristic transcription factors, including C/ebpβ, important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human confirmed high molecular similarity in immature stages and discriminated zebrafish-specific from pan-species gene signatures. Applying pan-species neutrophil maturation signatures in RNA-seq data from neuroblastoma patients revealed an association of metastatic tumor cell infiltration in the bone marrow with an increase in mature neutrophils. Our detailed neutrophil maturation atlas provides a valuable resource for studying neutrophil function at different stages across species in health and disease. Graphical

In this webinar, we will give an overview of imaging-based spatial transcriptomics (IST) technologies and discuss how preprocessing can affect downstream analysis results. Using our recently developed QC package (Huber group, EMBL), we will demonstrate a workflow for evaluating segmentation quality and identifying transcript assignment errors in IST data.