Bone Marrow-Infiltrating Human Neuroblastoma Cells Express High Levels of Calprotectin and HLA-G Proteins

Fabio Morandi,Fabio Gallo,Paola Scaruffi,Giampaolo Arcamone,Stefano Bonassi,Katia Mazzocco,Riccardo Haupt,Stefano Moretti,Sara Stigliani,Gian Paolo Tonini,Vito Pistoia,Maria Valeria Corrias,On Behalf Of The Italian Cooperative Group For Neuroblastoma ,Claudio Gambini,Paolo Fardin

doi:10.1371/journal.pone.0029922

Abstract

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. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. 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.

Highlights

Metastasis is the main cause of death in patients with neuroblastoma (NB) [1,2,3]
After in vitro culture of bone marrow (BM) samples from patients with metastatic disease, Hansford et al [13] isolated cells endowed with high tumorigenic potential, suggesting that metastatic cells may be enriched in tumor initiating cells (TICs)
Characterization of BM-infiltrating GD2 positive cells To ascertain whether genes expressed by the GD2 positive fractions can be fully ascribed to the BM-infiltrating NB cells, six freshly isolated GD2 positive cell populations were characterized using different methodologies

Summary

Introduction

Metastasis is the main cause of death in patients with neuroblastoma (NB) [1,2,3]. In spite of aggressive multimodal therapy, children over 18 months of age, presenting at diagnosis with dissemination of malignant neuroblasts in the bone marrow (BM), have a grim prognosis [1,2]. To achieve efficient targetbased therapies for high risk NB patients, characterization of the genetic and biological features of metastatic cells is required. Extensive characterization of primary tumor cells from high-risk NB patients has been performed. A great effort has been performed in order to validate the gene classifiers on independent patient cohorts [7,10]. After in vitro culture of BM samples from patients with metastatic disease, Hansford et al [13] isolated cells endowed with high tumorigenic potential, suggesting that metastatic cells may be enriched in tumor initiating cells (TICs). A gene expression profiling of TICs has been reported [14]. The proteins selectively overexpressed by the BM-infiltrating NB cells may represent novel prognostic markers and potential targets for biologically driven therapy for high risk NB patients

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