(c), FISH analysis with chromosome 17q-specific probe on MELC-preprocessed sample BM 1.1, collected from a patient with 17q gain. surrounding cells, i.e., the microenvironment, in bone marrow metastases, using the childhood tumor neuroblastoma as a model. To this end, we screened genome-wide datasets to define a panel of cell-specific markers for multiplex microscopy of metastatic bone marrow samples, and developed DeepFLEX, a computational pipeline for subsequent image analysis. Thereby, we identified 35,000 single cells covering metastasized tumor cells, and various types of developing immune and bone marrow cells. In parallel, we analyzed the transcriptome, i.e., all genes that are expressed as mRNA, of 38 patients with and without bone marrow metastasis. We found vast tumor cell diversity and identified a marker protein, FAIM2, which can gamma-secretase modulator 2 help to identify a broader range of tumor cell variants. In addition we showed that tumor cell metastasis in the bone marrow is associated with an immune response resembling inflammation, and the presence of cells that can repress an immune attack against cancer cells. Our study suggests that metastatic tumor cells are shaping the bone marrow microenvironment and builds the basis to further investigate its clinical relevance. Abstract 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 gamma-secretase modulator 2 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 usually 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. Rabbit Polyclonal to Cyclin D3 (phospho-Thr283) amplification status for all those neuroblastoma cell lines were described previously [37] and are listed in Table S1. Cells were maintained in RPMI1640-Glutamax-I (GIBCO) supplemented with 1% Pen/Strep (GIBCO), 10% FCS (PAA Laboratories), 1 mM sodium pyruvate (PAN Biotech) and 25 mM HEPES (PAN Biotech). All neuroblastoma cell lines were cultivated at 37 C and 5% CO2. 2.1.2. Bone Marrow Aspirates Bilateral bone marrow aspirates were collected according to the SIOPEN/HR-NBL-1 study protocol or standard of care during routine diagnostics at initial diagnosis and at clinical response evaluation time points. Samples were shipped at room temperature within 4 h or at 4 C within 24 h. Bone marrow-derived mononuclear cells (MNCs) were isolated by density gradient centrifugation (LymphoprepTM, AXIS-SHIELD PoC AS). For the validation of antibodies, gamma-secretase modulator 2 neuroblastoma cell lines were mixed with tumor-free bone marrow-derived MNCs to obtain a tumor cell suspension of 5% neuroblastoma cell line in bone marrow-derived MNCs. For single-cell analysis, eight bone marrow aspirates (Table S2) were collected at different time points along the therapy protocol from four neuroblastoma patients with metastatic (INRG stage M or Ms) disease. For bulk RNA-sequencing for this study 38 bone marrow aspirates from 38 patients.