Gene expression analisys of GD2+ cells from metastatic NB, unselected bone marrow from metastatic NB, primary tumors from stage 4 NB (alive and dead at 5 years of follow-up) and primary tumor from localized NB

Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Metastatic NB cells were freshly isolated from patients’ BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Unselected BM samples from patients with metastatic NB were also included. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients. This is an update of the previous GEO submission (GSE25623). We performed a gene expression analysis on 57 samples: 11 GD2 positive cells from metastatic NB, 9 bone marrow from metastatic NB, 13 primary tumors from stage 4 NB alive at 5 years of follow-up, 14 primary tumors from stage 4 NB dead at 5 years of follow-up, and 10 primary tumors from NB patients with localized disease