MIF/CXCR4 signaling axis contributes to survival, invasion, and drug resistance of metastatic neuroblastoma cells in the bone marrow microenvironment

Abstract

Background: The bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets.

Methods: Patient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O₂) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo.

Results: We identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model.

Conclusions: Our findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB.

Keywords: 4-IPP; Bone marrow; CXCR4; Hypoxia; MIF; Neuroblastoma.

Fig. 1

Generation and characterization of CMs mimicking the BM niche. A) Quantification of different cell populations in expanded BM cultures (n = 6) by flow cytometry (empty dot plots). Purified culture of BM-MSC (column bars) was used as positive control for stroma markers (CD90, CD105 and HLA-DR). B) Graphical scheme of CM’s production (left). In vitro image (10x objective) of cells used for CM’s production at 48 h (right). C) Cytokine Profile Array membranes of CM-NB, CM-BM, CM-BM/NB (left). Quantification of detected cytokines and chemokines represented as mean pixel density of duplicate spots (right). D) Extracellular MIF quantification by ELISA in CM-NB (n = 6), CM-BM (n = 5) and CM-BM/NB (n = 6), being LAN-1 square, SH-SY5Y rhomboid, and IMR5 a circle

Fig. 2

MIF and CXCR4 are expressed in high-risk NB tumors and cell lines. Violin plots showing CXCR4, CD74, MIF and CXCL12 gene expression across A) different NB stages and B) risk groups, low/intermediate-risk (LIR) and High-risk (HR) (GSE62564). *P < 0.01 (One-way ANOVA, Kruskal Wallis test). C) NB patient overall survival associated with CXCR4, CXCL12, CD74 and MIF expression levels (GSE62564, Kaplan-Meier analysis). High expression is shown in green and low expression in red. Total number of patients and (positive events) per group are also shown. Percentage of D) CXCR4 and E) CD74 positive cells in a NB cell line panel analyzed by flow cytometry (n = 3)

Fig. 3

BM-like culture conditions promote NB proliferation, invasion, and drug resistance. A) LAN-1, SH-SY5Y, IMR5, and HSJD-NB01 cells were cultured in CMs under normoxic, and hypoxic conditions and MTS assay was performed over 4 days. Data are represented as a fold change viability to time 0 h (n = 6). B) Wound healing assay of LAN-1 cells after 24 h (left) and its quantification (right). Differences were calculated comparing each experimental condition to control (CM-CNT in normoxia) (n = 3). Normoxia conditions (black) hypoxia conditions (blue). Two-way Anova, Dunnett’s multiple comparison test. C) Transwell invasion assay of LAN-1 cells after 96 h (left) and its quantification (right). Differences were calculated comparing each experimental condition to control (CM-CNT in normoxia) (n = 3). Two-way Anova, Dunnett’s multiple comparison test. D) Activity of chemotherapy (doxorubicin, etoposide, and SN-38) over LAN-1 cells cultured with CM-BM/NB at 72 h (n = 3). Normoxia conditions (black) hypoxia conditions (blue). (*) P < 0.05

Fig. 4

In vivo effects of BM in a LAN-1 xenograft model: A) Engraftment curves for each group, reported at 100 mm³ tumor size. B) Tumor survival curves for each group, reported at 1500 mm³ tumor size. C) Histology of primary subcutaneous LAN-1 tumors by H&E, hNu, and Ki-67 staining (40x). D) Ki-67 quantification of stained FFPE sections (n = 6) was quantified using ImageJ software. Mann-Whitney test. (*) P < 0.05

Fig. 5

BM-like culture conditions increase CXCR4 surface expression and activate AKT and ERK pathways. A) Flow cytometry histograms of CXCR4 expression in LAN-1 cells cultured under BM-like conditions for 72 h (left). CXCR4 expression is represented as a fold-change to CM-CNT Nx (n = 3; right). B) Flow cytometry histograms of CXCR4 expression in SH-SY5Y cells cultured under BM-like conditions for 72 h (left). CXCR4 expression is represented as a fold-change to CM-CNT Nx (n = 3; right). Histograms were generated with NovoFlow Software (Acea Bioscience). Normoxia results are in black and hypoxia in blue. (*) P < 0.01 (Two-way ANOVA, Dunnett’s multiple comparison test). Immunoblots of LAN-1 C) and SH-SY5Y D) cultured under BM-like conditions for 48 h. Band intensity p-AKT (S473), p-ERK (T202/Y204 T185/Y187) ratios are calculated by measuring the relative protein levels (phosphorylated/total), and γ-Tubulin (as a loading control) by using ImageJ Software (n = 2)

Fig. 6

The activity of MIF inhibitors on neuroblastoma cell lines and BM primary cultures. Cytotoxic activity of A) AMD3100 (50-0.0001 μM), B) ISO-1 (50-0.2 μM) and C) 4-IPP (50-0.2 μM) in a panel NB cell lines and BM primary culture under regular conditions (n = 6). Flow cytometry quantification in bar graphs of 4-IPP activity on D) BM, E) LAN-1, and F) co-cultures LAN1:BM ratio (1:10) treated with vehicle or 4-IPP 30 μM after 72 h at regular conditions (n = 2)

Fig. 7

Impairing NB aggressive phenotypes with MIF and CXCR4 inhibitors. A) Cell viability after exposure to CM-CNT or CM-BM/NB and treatments (vehicle, 10 nM AMD3100 and 5 μM 4-IPP) at 72 h. Data is represented as a fold-change viability to time 0 h (n = 3). Significances were obtained after comparing vehicle vs treatments. One-way Anova Kruskal-Wallis test. B) Images (10x) and graph bar quantification of LAN-1 wound healing recovery under CM-CNT and CM-BM/NB normoxia and hypoxia (vehicle, 10 nM AMD3100 and 5 μM 4-IPP) at 24 h. Two-way Anova Sidaks’s multiple comparison test. C) Images and graph bars of invaded cells with 5 μM 4-IPP or 10 nM AMD3100, under CM-BM/NB normoxia (black) and hypoxia (blue) at 96 h. One-way Anova Kruskal-Wallis test. * P < 0.05. D) Cytotoxicity of chemotherapy compounds, doxorubicin and etoposide, in LAN-1 cells cultured with CM-BM/NB at 72 h together with 5 μM of 4-IPP or vehicle. Bar graph with relative viability at CM-BM/NB for doxorubicin and etoposide IC50. Normoxia (black), hypoxia (blue). Two-way Anova, Dunnett’s multiple comparison test. (n = 3) (*) P < 0.05. E) Individual tumor growth in vehicle and 4-IPP treated group. F) Tumor survival curves for each group. Events were reported when tumor size reached 1000 mm³. G) Body weight variation of mice during treatment (grey area) and follow-up