Neuroblastoma and bone marrow cell cultures
Neuroblastoma (NB) cells LAN-1 (MYCN amplified, ALK F1174L, origin BM), SH-SY5Y (MYCN non-amplified, ALK F1174L, origin BM), IMR5 (MYCN amplified, ALK WT, origin abdominal mass), and the patient-derived cell line HSJD-NB01 (stage 4, MYCN amplified, origin suprarenal mass) were obtained from the repository of the Laboratory of Developmental Tumors, Hospital Sant Joan de Déu (HSJD), Barcelona and previously characterized in [22]. Cell cultures were maintained in regular culture conditions: RPMI-1640 supplemented with 10% of inactivated Fetal Bovine Serum (iFBS), 2 mM L-glutamine, and Penicillin/Streptomycin (100 μg/mL and 100 μg/mL, respectively) (Thermo Fisher Scientific, USA) in a controlled atmosphere at 37 °C and 5% CO2. Cell culture experiments were performed either at atmospheric oxygen levels [normoxia (Nx); 21% O2] or [hypoxia (Hx); 1% O2]. Oxygen levels were controlled with a modular incubator chamber MIC-101 (Billups-Rothenberg, Inc., USA) and cobalt chloride (CoCl2, Sigma-Aldrich, USA) was used as hypoxia-state control.
To establish patient-derived BM primary cultures, fresh BM aspirates were obtained from 20 NB patients treated in our institution. Samples were obtained at relapse or during consolidation therapy for NB (Additional Table 1). Samples were obtained under an Institutional Review Board approved protocol at Hospital Sant Joan de Déu, Barcelona, Spain. Parents and/or guardians signed informed consent before the collection of samples.
BM aspirates were collected and centrifuged in a density gradient Histopaque®-1077 (Sigma-Aldrich) at 2000 g for 20 minutes to obtain BM mononucleated cells. Cells were then plated and expanded in regular culture conditions up to 15 days before conditioned media generation. NB minimal residual disease (MRD) status within BM aspirates was detected by analysis of Paired-like Homeobox 2B gene (PHOX2B) gene expression using Real-Time Quantitative PCR, following previously described protocols [23] (Additional Table 1).
Conditioned media (CM)
To generate CMs, we used in vitro expanded BM cells (2 × 106; CM BM), BM cells co-cultured with NB cells in a ratio of 1:1 (1 × 106 each; CM-BM/NB) or NB cells cultured alone (2 × 106; CM-NB). After 48 hours in culture, CMs were collected, centrifuged at 1000 g for 10 minutes and kept at − 80 °C for future assays. Before each experiment, CMs were thawed and diluted 1:1 with regular RPMI without iFBS. As a control condition, CM-CNT was composed of complete RPMI-1640 with 5% of iFBS.
Genomic data
Two publicly available RNA sequencing (RNA-seq) data sets were used for the study. GSE94035 comprised RNA-seq data from 16 primary tumors, 42 BM-derived disseminated NB tumor cells and corresponding 28 BM-derived non-tumor cells of NB patients [12], and GSE62564 comprised 498 primary NB diagnostic biopsies, including INSS (International Neuroblastoma Staging System) stage 1-3 and 4S as well as stage 4 NB specimens. In addition, GSE62564 data set were analyzed according to the classification criteria of High-Risk NB Study 1.7 of SIOPEurope (SIOPEN) (NCT01704716). Nine samples were not included in the study due to a lack of available clinical information [24]. The Contal-O’Quigley method was used to establish the threshold for overall survival (OS) studies (Kaplan-Meier method) [25]. The following thresholds were obtained: MIF (7.1; log-rank p < 0.0001), CXCL12 (3.5; log-rank p < 0.0001), CXCR4 (5.9; log-rank p = 0.00018), CD74 (7.4; log-rank p < 0.0001), CD44 (7.7; log-rank p < 0.0001), CXCR7 (2.9; log-rank p = 0.59), and CXCR2 (0.8; log-rank p = 0.018). The log-rank test was used to analyze survival differences performed with GraphPad Prism 8 (La Jolla, USA).
MIF and CXCR4 inhibitors
AMD-3100 (Plerixafor), ISO-1 (Selleckchem, Germany), and 4-Iodo-6-phenylpyrimidine (4-IPP) (Merck, Germany) were diluted with ethanol at a stock concentration of 10 mM. Cell viability was assessed at different concentration ranges using MTS assay (Promega Inc., USA). NB cell lines and fresh BM-MNCs were cultured in 96-well plates for 24 hours in regular media before being treated with drugs at concentration ranges: AMD-3100 (50-0.0001 μM), ISO-1 (50-0.2 μM), 4-IPP (50-0.2 μM) (n = 6 per each concentration). After 72 hours, cell viability was compared to control wells (vehicle 100% viable). The half-maximal inhibitory concentration (IC50) of tumor cell viability was calculated with a nonlinear regression log (inhibition) vs. response variable slope using the software Prism 8 (GraphPad, USA).
Cell viability in bone marrow-based in vitro conditions
To test CM-driven changes in NB cell viability, LAN-1 and SH-SY5Y (2 × 103 cells per well), HSJD-NB01 (8 × 103 cells per well) and IMR5 (8 × 103 cells per well) cell lines were plated in 96-well plates with 100 μL of regular media for 24 h. Regular culture media was then replaced by CM-NB, CM-BM, CM-BM/NB, CM-CNT and cultured under normoxic or hypoxic conditions (n = 6 replicates per condition). Cell viability was measured with MTS assay (Promega) at 0, 24, 48, 72 and, 96 h at 490 nm with microplate reader Infinite® 200 PRO (Tecan, Switzerland). Cell viability was represented as a relative fold-change compared to the initial MTS signal at time 0 h. To assess whether treatments could affect NB cell viability in the presence of CMs, the same protocol was performed adding 5 μM of 4-IPP, 10 nM of AMD3100, 10 ng/mL recombinant human MIF (SRP3321, Millipore Sigma) or vehicle.
siRNA mediated knockdown
Human CXCR4 gene was transiently silenced in LAN-1 and SH-SY5Y neuroblastoma cells using Dharmacon ON-Targetplus Human CXCR4 siRNA technology (Horizon Discovery, USA). Cells were transfected with Lipofectamine® RNAiMAx transfection Reagent (Invitrogen, USA) and a 10 nM mixture of SMARTpool siRNA against human CXCR4 or a ON-TARGETplus Non-targeting Control Pool, following manufacturer’s indications.
Cell migration and chemoattraction assays
NB cells (8 × 105 cells per well) were plated into 6-well plates and allowed to reach a confluent monolayer. The monolayer was scratched with a pipette tip, and regular cell culture media was replaced by CM and incubated in normoxic or hypoxic conditions for 24 h. Cells were also treated with 5 μM of 4-IPP or 10 nM of AMD3100 in CM-BM/NB. After 24 h, the percentage of the cell-free area was measured with ImageJ Plugin Wound Healing Tool by RIO Imaging. Cell chemoattraction and invasion were tested using 8.0 μM pore membrane Cell Culture Insert Transwells (Corning, USA) pre-coated with 10% of Matrigel (Corning, USA) in RPMI media. Transwell upper chambers were filled with 2 × 105 cells per chamber in 200 μL of regular media with 1% iFBS, whereas lower chambers were filled with 500 μL of each CM and incubated in normoxic and hypoxic conditions for 96 h. Finally, invaded cells were fixed and stained with 4% paraformaldehyde for 10 min and 2% crystal violet for 30 min. In experiments using inhibitors drugs, cells were pre-treated overnight with 5 μM of 4-IPP or 10 nM of AMD3100 and then plated maintaining drug treatment in Transwell’s upper chambers. The surface area invaded by NB cells was measured in three representative areas per Transwell membrane using light microscopy (20x; DM 5000 B Leica, Germany) with ImageJ Software [26].
Drug response studies
LAN-1 cells (2 × 103 cells per well) were plated in 100 μL of CM-CNT or CM-BM/NB under normoxia or hypoxia for 24 h. Drugs were added at different concentration ranges: doxorubicin (2500-0.381 nM), etoposide (3750-0.572 nM), and SN-38 (500-0.076 nM). After 72 h, an MTS assay (Promega) was performed and IC50 concentrations were determined for each different drug and culture condition. To assess whether drug response could be modulated after MIF inhibition, the same protocol was performed adding to each experimental condition 5 μM of 4-IPP, or vehicle together with chemotherapeutic agents. Results are expressed as the percentage of relative viability, in which each experimental conditions were compared to their own control.
RNA extraction and RT-qPCR
Total RNA was isolated using TRI Reagent (Sigma-Aldrich) and quantified with NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was generated with M-MLV reverse transcriptase system (Thermo Fisher Scientific) using 1 μg of total RNA as previously reported [27]. Gene expression was quantified using SYBR® Green PCR Master Mix in a QuantStudio 6 Real-Time PCR system (Thermo Fisher Scientific). Sequences and references for primers are listed in Additional Table 2. Relative expression of mRNA was determined using the 2-(ΔΔCt) quantification method and normalized to YWHAZ as a reference gene [28].
Proteome arrays
Determination of cytokine-expression levels was performed using Proteome Profiler Human Cytokine Array Kits (R&D Systems, USA) following the manufacturer’s protocols. The chemiluminescent signal was detected with the iBright Imaging System (Thermo Fisher Scientific) and quantified with QuickSpots Software (R&D Systems).
Enzyme-linked immunosorbent assay (ELISA)
CXCL12 and MIF secretion in CM was measured using the Human CXCL12/SDF-1 DuoSet ELISA and MIF Quantikine ELISA kits (R&D Systems) following the manufacturer’s protocols. The absorbance was measured at 450 nm and corrected at 540 nm on a microplate reader Infinite® 200 PRO (Tecan, Switzerland).
Immunoblotting
Cell samples were lysed with RIPA 1X buffer (50 mM Tris pH 8.8, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% NP40) plus complete Proteases Inhibitor Cocktail Tablets (Roche, Switzerland). Protein extracts were quantified with Bradford Reagent (BioRad, USA). Samples were separated by electrophoresis in polyacrylamide gels (8 - 12%), transferred to nitrocellulose membranes (Thermo Fisher Scientific) and PageRuler™ Plus Prestained Protein Ladder as protein size control (Thermo Fisher Scientific). Immunoblot detection was performed with near-infrared labeled secondary antibodies in the imaging system Odyssey® CLx (LI-COR Inc., USA) and software Image Studio® (LI-COR Inc., USA) following manufacturer’s instructions. Primary and secondary antibodies are listed in Additional Table 3. Relative protein levels were measured, and ratios calculated (phosphorylated/total) by ImageJ Software.
Flow cytometry
Cell cultures were washed in blocking-PBS buffer [1% Bovine Serum Albumin (Sigma-Aldrich) and 1% FBS in PBS 1X (Thermo Fisher Scientific)], stained with primary-conjugated antibodies (Additional Table 3), as well as with 0.2 μg/mL 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) to select viable cells. Flow cytometry data were analyzed with ACEA Novocyte 3000 (Acea Bioscience Inc., USA), and results were processed with NovoExpress Software (Acea Bioscience Inc.)
BM characterization
On day 15 of BM expansion, BM cultures (n = 6) were washed and stained with two panels of fluorochrome-conjugated antibodies (BD Bioscience, USA). A primary culture from non-oncologic BM-derived mesenchymal stem cells (BM-MSCs) was used as a control sample for mesenchymal markers. Panels: Panel 1 included CD11b-PE, CD105-FITC, CD45-V500, CD34-PerCP, CD19-PeCy7, and CD123-APC. Panel 2 included CD90-PE, CD45-V500, HLA-DR-PerCP, CD19-PeCy7, and CD117-APC. The percentages of mesenchymal cells were calculated as CD45−/CD90+/HLA-DR+ and CD45−/CD105+, whereas percentages for the rest of markers were defined as positive stained events over the CD45+ population. Sample acquisition was performed in a FACSCanto II (BD Bioscience, USA) and all channels were previously compensated for fluorescence spillover.
CXCR4 and CD74 expression
NB cells cultured with CM-CNT, CM-NB, CM-BM, CM-BM/NB, under normoxic or hypoxic conditions were collected with Versene (Thermo Fisher Scientific) at 72 h and exposed to primary-conjugated antibodies for 30 min at room temperature protected from light exposure. Basal cell surface expression of CXCR4 and CD74 was measured in LAN-1, SH-SY5Y and IMR-5 cells under regular culture conditions using the same method.
In vitro co-culture studies
Co-cultures combining NB and fresh BM-MNCs cells were performed to evaluate the specific vulnerability of tumor cell lines but not BM cells to MIF inhibition. Thus, NB cells were plated in 6-well plates (5 × 103 cells per well) for 24 h in regular media. Then, BM cells were added in a ratio of 10:1 (BM:NB) together with 30 μM of 4-IPP or vehicle, in regular media. NB and BM cells cultured alone were used as positive and negative controls, respectively. After 72 h, floating and attached cells were collected, washed, and stained with primary-conjugated antibody for 30 min at room temperature avoiding light exposure.
In vivo assays
Animal procedures were performed at the animal facility at HSJD according to regulatory order (214/97, Generalitat de Catalunya) and the local animal ethics committee (9330 CEEA, Universitat de Barcelona). Research with mice adhered to European regulations and ARRIVE guidelines and approved by the animal experimental Ethics Committee of the Universtidad de Barcelona and the Generalitat de Catalunya (9330 CEEA; animal protocol number 214/97).
Xenograft models were generated in 5-6 weeks old female athymic nude mice (Envigo, Spain). LAN-1 cells were injected subcutaneously (s.c.) at both flanks in a mixture of Matrigel® and RPMI medium (vol/vol). Every 3-4 days animals were followed up by measuring weight and tumor size with a caliper, as well as monitored for macroscopic metastasis events. Tumor size (mm3) was calculated under the formula: 1/2(length × width2) [29]. Endpoint criteria were set when tumor size reached ≥1500 mm3 at a single flank or animals lost 20% of the weight. Engraftment and survival analyses were calculated by Kaplan-Meier Method performed with Prism 8 software (GraphPad, USA).
Tumor growth under stromal support
LAN-1 cells were injected s.c. alone (5 × 105 cells; NB group; n = 18) or together with BM expanded cells in a 1:1 ratio (5 × 105 each cell type; BM/NB group; n = 22) to compare tumor engraftment (tumor size ≥100 mm3), progression, and survival. As a negative control, a small group of mice was injected with BM cells alone (group BM, n = 4). Data gathered from two different in vivo experiments are represented together. Tumor masses were harvested and fixed in 4% formaldehyde solution for posterior immunohistochemistry (IHC) studies.
In vivo treatment
Antitumor efficacy of the MIF inhibitor 4-IPP (Merck) was tested in LAN-1 xenografts. For in vivo treatment, 4-IPP was diluted in corn oil (Sigma-Aldrich) as previously described [30]. LAN-1 cells (1.5 × 106 cells) were injected s.c. in both flanks and 7 days later mice were randomly distributed (vehicle, n = 14) and treatment (4-IPP, n = 10) groups. 4-IPP treatment was administrated via intraperitoneal route 3 days a week every 2 days for 4 weeks at 80 mg/kg.
Immunohistochemistry
After fixation, tissues were embedded in paraffin by conventional automated systems. Four-micron sections were stained with hematoxylin-eosin (H/E), human nuclei antibody, and Ki-67 (Additional Table 3).
Statistical analyses
Unless otherwise specified, group differences were calculated comparing each experimental condition to control (CM-CNT under normoxia) with Two-way ANOVA and Dunnett’s multiple comparison test. Survival curves were estimated by Kaplan-Meier method and compared among groups by log-rank test. Statistical analysis was performed with the software Prism 8 (Graphpad, USA). Significance was defined as P < 0.05 or P < 0.01.