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. 2023 May 4;14:2575. doi: 10.1038/s41467-023-38239-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — NOR noradrenergic, MES mesenchymal. A Ex vivo culture of the noradrenergic IC-pPDX-63 neuroblastoma model established from a brain metastasis of a stage 4 case at relapse resulted in the IC-pPDXC-63 cell line that includes floating neurospheres and adherent cells as observed by contrast phase microscopy. B Heatmap and unsupervised clustering of samples (cell lines, patient tumors and PDXs) using the expression of transcription factors (TFs) of the noradrenergic and mesenchymal identities^17,18 on bulk RNAseq data. The transcriptomic profile of the IC-pPDX-63 model (bulk RNAseq of the PDX tumor (PDX) or from its single-cell (scPDX)) and its derived-cell line (CL_IC-pPDXC-63) are noradrenergic and highly similar to that of the matched patient tumor (IC-63) from which it has been generated. Two replicates of CD44^pos and CD44^neg cell sorts of the cell line are included (a and b). CL cell line, flo floating neurospheres, adh adherent cells. hNCC human neural crest cells. Source data are provided as a Source Data file. C Single-cell transcriptomic analyses of the IC-pPDXC-63 cell line by Seurat showing clustering at resolution 0.8, cell cycle phases, and the noradrenergic and mesenchymal identities highlighted by noradrenergic and mesenchymal transcription factor signatures^17,18 and PHOX2B and CD44 expression, respectively, plus a bridge in-between. D Immunofluorescence shows the specific expression of the PHOX2B and CD44 markers by neurospheres and adherent cells, respectively (scale bar = 20 µm, representative of 3 independent experiments). E Inferred genomic profile of the IC-pPDXC-63 cell line obtained with InferCNV on single-cell data. F Mesenchymal (IC-pPDXC-63 CD44^pos) cells are more resistant to chemotherapy than noradrenergic (IC-pPDXC-63 CD44^neg) cells. Cell viability was measured with resazurin assay after 72 h of chemotherapy treatments (Doxorubicin 50, 100, 250 nM and Etoposide 0.5, 1, 2.5 µM, mean ± sd; n = 6 replicates). P-values were determined via two-tailed unpaired Welch’s t-test (***p < 0.000001). Source data are provided as a Source Data file.