Fig 2: Characterization of DTC heterogeneity and qualification of FAIM2 as a novel complementary biomarker. (a), Cluster map (hierarchical clustering by Voorhees) showing normalized single-cell feature values of DTCs. n, nucleus; c, cell. Nine columns per marker represent, from right to left, mean intensity, total intensity and mean of the highest 20% of pixel values in the (I) nucleus, (II) cell and (III) cytoplasm/membrane. Color bar on the right shows 30 sub-clusters. Color bar on the left shows the corresponding bone marrow sample. (b), Representative gallery images of six selected cells from different DTC sub-clusters reflecting DTC heterogeneity. For FAIM2 we introduced negative controls (NC) to be used as background threshold levels during data processing. Hence, for this biomarker, the ratio between right column and left column (NC) represents the true signal. (c), Proportion of 30 DTC sub-clusters in highly tumor-infiltrated bone marrow samples (BM 1.1, BM 3.1). (d), CD56 and CD276 mRNA transcription in bone marrow-derived mononuclear cells (MNCs) and neuroblastoma tumor cells (TU/DTC) without (wt) and with (amp/loss) genetic aberration. Wilcoxon–Mann–Whitney with FDR-corrected p-values: ns, **, p = 0.01; ****, p = 0.0001. FPM, fragments per million; amp, amplification.

Fig 3: Data mining and establishment of a multiplex imaging panel (MELC). (a), Flow chart of experimental approach. (b), five potential disseminated tumor cell (DTC) biomarkers identified by data mining of RNA-seq data, proteomics data (LC-MS/MS) and literature. Top: mRNA transcription (RNA-seq) in neuroblastoma primary tumors (TU), diagnostic (dx) and relapse (rel) DTCs and bone marrow-derived mononuclear cells (BM-MNCs). DESeq2, FDR-adjusted p value: ns, p > 0.05, *, p = 0.05; **, p = 0.01; ***, p = 0.001. Bottom: Protein expression in NB peripheral-nerve-associated fibroblasts (PF), neuroblastoma cells lines and TU samples. LFQ, Label-Free Quantification; FPM, fragments per million. (c), Extension of potential DTC biomarkers by immune checkpoint molecules (PD-L1, PD-1, B7-H3), mesenchymal-type neuroblastoma cell markers (VIM, PROM1), therapeutic target NCAM-L1 and diagnostic neuroblastoma marker CD56. DESeq2, FDR-adjusted p value: ns, p > 0.05, *, p = 0.05; **, p = 0.01; ***, p = 0.001. (d), Representative MELC images of newly identified DTC biomarkers DCLK1, FAIM2 and TAG1 on separate samples stained by MELC. Top: DCLK1 (green) and GD2 (red) on BM-MNCs and neuroblastoma cells line STA-NB-10 (mixed 20:1); center: FAIM2 (green) and GD2 (red) on BM-MNCs and neuroblastoma cell line STA-NB-2 (20:1), bottom: TAG1 (green) on peripheral blood-derived MNCs and neuroblastoma cell line STA-NB-4 (20:1) stimulated with IFN? and anti-CD3/28 beads. Nuclei were counterstained with DAPI (blue). (e), Representative MELC images of our single-cell 20-plex panel on one patient bone marrow sample. (f), Single-cell 20-plex panel composed of DTC, myeloid, lymphoid, mesenchymal and HSPC (hematopoietic stem and progenitor cell) markers.