Mesenchymal stromal cell secretome up-regulates 47 kDa CXCR4 expression, and induce invasiveness in neuroblastoma cell lines

Abstract

Neuroblastoma accounts for 15% of childhood cancer deaths and presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Previous studies have shown that the Mesenchymal Stromal Cell (MSC) secretome, triggers metastases in several cancer types such as breast and prostate cancer, but the specific role of the MSC factors in neuroblastoma metastasis is unclear. To better understand the effect of MSC secretome on chemokine receptors in neuroblastoma, and its role in metastasis, we studied a panel of 20 neuroblastoma cell lines, and compared their invasive potential towards MSC-conditioned-RPMI (mRPMI) and their cytokine receptor expression profiles. Western blot analysis revealed the expression of multiple CXCR4 isoforms in neuroblastoma cells. Among the five major isoforms, the expression of the 47 kDa isoform showed significant correlation with high invasiveness. Pretreatment with mRPMI up-regulated the expression of the 47 kDa CXCR4 isoform and also increased MMP-9 secretion, expression of integrin α3 and integrin β1, and the invasive potential of the cell; while blocking CXCR4 either with AMD 3100, a CXCR4 antagonist, or with an anti-47 kDa CXCR4 neutralizing antibody decreased the secretion of MMP-9, the expression of integrin α3 and integrin β1, and the invasive potential of the cell. Pretreatment with mRPMI also protected the 47 kDa CXCR4 isoform from ubiquitination and subsequent degradation. Our data suggest a modulatory role of the MSC secretome on the expression of the 47 kDa CXCR4 isoform and invasion potential of the neuroblastoma cells to the bone marrow.

Fig 1. Invasive potential of neuroblastoma cell lines.

a. 20 neuroblastoma cell lines were serum-starved for 24 hours, and plated at a seeding density of 0.5 X 10⁵ cells per well on to 0.8 micron cell culture inserts, coated with Basement Membrane Extract (BME) coat at 0.5x to 1x dilution. The number of cells crossing the mesh towards RPMI (control) or mRPMI were counted fluorimetrically using calcein AM after 12 hours. Plotted is the average number of cells, for each cell line from 7 experiments, migrating across the mesh. The red line indicates the mean value. Cell lines higher than the mean value were classified as highly invasive and those below the mean value as less invasive cell lines. b. Invasion index was calculated as the ratio of number of cells invading towards mRPMI versus the number of cells invading towards RPMI. Plotted is the mean invasion index from 7 experiments in increasing order. Cell lines with an invasion index of 1 or higher was classified as highly invasive cell lines and those with an invasion index of less than 1 was classified as less invasive.

Fig 2. Expression of cytokine receptors CXCR4, CXCR5 and CXCR6 in neuroblastoma cell lines.

a. Cell surface expression of CXCR4 on neuroblastoma cell lines was assayed by flow cytometry. Cells were harvested using trypsin, washed and incubated with PE-conjugated anti-CXCR4 antibody. After 30 minutes of incubation, the cells were washed and analyzed on a BD FACSCaliber flow cytometer using CellQuest. One thousand events were counted for each sample and the mean fluorescent intensity (MFI) from three experiments were calculated. Plotted is the MFI in increasing order. b. Plotted are the relative total CXCR4 expressions (MFI from 3 experiments) (y axis) and the invasion indices (x axis) of 20 neuroblastoma cell lines. c. Surface expression of CXCR5, plotted as MFI from three experiments. d. Surface expression of CXCR6, plotted as MFI from three experiments.

Fig 3. Structural heterogeneity of CXCR4 in neuroblastoma cell lines.

a. Western blot detection of CXCR4 in 20 neuroblastoma cell lysates. 1 X 10⁷ cells were washed twice in PBS and solubilized in 1 ml of lysis buffer. The lysates were centrifuged at 14,000 rpm at 4°C for 30 minutes. Equal amounts of total proteins, as determined by BCA assay, were loaded on to a 12.5% polyacrylamide gel and electrophoresed. The proteins were then blotted on to a PVDF membrane and CXCR4 was detected using an anti-CXCR4 polyclonal antibody and a HRP conjugated secondary antibody. Shown in figure is a representative result from 5 experiments. b. The bands from western blot studies were quantified using imageJ. Plotted is the mean relative expression of CXCR4 isoforms in the whole cell lyaste from 5 experiments. c. CXCR4 was immune precipitated using the Pierce Crosslink Immunoprecipitation kit with anti-CXCR4 antibody. The precipitate was electrophoresed and blotted on to PVDF membrane. Protein detection was carried out using a second anti-CXCR4 antibody. Shown in figure is a representative result from 5 experiments. d. The bands from western blot studies were quantified using imageJ. Plotted is the mean relative expression of CXCR4 isoforms in the CXCR4 immune precipitate from 5 experiments.

Fig 4. Localization and CXCL12 reactivity of CXCR4.

a. The membrane proteins from 3 highly invasive neuroblastoma cell lines, MNB-OZ, NB 19 and SK-N-SH, and 2 less invasive cell lines, IMR 32 and SJ-N-KP were extracted, electrophoresed and blotted on to PVDF membrane. Protein detection was carried out using an anti-CXCR4 polyclonal antibody. Shown in figure is the representative result from 5 experiments. b. CXCR4 immune-precipiate from 3 highly invasive (NB 19, MNB-OZ & SK-N-SH) and 3 less invasive (SJ-N-KP, IMR 32 & NB Mass) cell lines were electrophoresed and blotted on to PVDF membrane. The membrane was incubated overnight with human CXCL12 in TBS-T at 4°C. The blot was washed and protein detection was carried out with anti-CXCL12 antibody. Shown in figure is the representative result from 3 experiments.

Fig 5. CXCR4 blocking assays.

a. Total cell lysate from MNB-OZ, NB 19 and SK-N-SH cells were electrophoresed and blotted on to PVDF membrane, and protein detection was carried out with anti-47 kDa neutralizing antibody. Shown in figure is the representative result from 5 experiments. b. 80% confluent SK-N-SH cells were harvested and incubated in either RPMI, RPMI + IgG control, RPMI + AMD 3100 or RPMI + anti-47 kDa neutralizing antibody. The supernatants were harvested after 24 hours, centrifuged and subjected to gelatin zymography. The gels were analyzed using imageJ and the relative MMP-9 secretion was calculated from 3 experiments (* p < 0.05). c. Total cell lysates from SK-N-SH cells either serum-starved (control), treated with IgG control (negative control), RPMI + AMD 3100 or RPMI + anti-47 kDa neutralizing antibody was subjected to western blot analysis. Protein detection was carried out with anti-integrin α3 or anti-integrin β1 antibodies. Anti-actin antibody was used for loading control. Shown in figure is the representative result from 3 experiments. d. Transwell invasion studies were carried out with cell lines SK-N-SH, MNB-OZ and NB 19. Cells were serum-starved (control), treated with 10 μM AMD 3100 and treated with anti-47kDa CXCR4 neutralizing antibody. Plotted is the mean number of cells migrating across the mesh. (* p<0.05) e. Invasion index of cell lines SK-N-SH, MNB-OZ and NB 19, serum-starved (control) and treated with AMD 3100 and anti-47 kDa CXCR4 neutralizing antibody, were analyzed using transwell invasion assays. Plotted is the normalized invasion index values obtained from 5 experiments (* p < 0.05).

Fig 6. Effect of MSC secretome on neuroblastoma cell lines.

a. (left panel) CXCR4 immune precipitates from cell lines MNB-OZ, NB 19 and SK-N-SH, serum-starved (control) and pre-treated with mRPMI were subjected to western blot analysis. Protein detection was carried out using anti-CXCR4 polyclonal antibody. Shown in figure is the representative result from 3 experiments. (right panel) CXCR4 immune precipitates from cell lines MNB-OZ, NB 19 and SK-N-SH, serum-starved (control) and pre-treated with mRPMI were electrophoresed and blotted on to PVDF membrane. The membrane was incubated with human CXCL12 overnight at 4°C. Protein detection was carried out using anti-CXCL12 antibody. Shown in figure is the representative result from 3 experiments. b. The blots were quantified using imageJ. Plotted are the mean relative expression of CXCR4 from 3 experiments. (* p < 0.05) c. Plotted are the mean relative CXCR4 bound CXCL12 from 3 experiments. (* p < 0.05). d. Culture supernatants from cells serum-starved (control) and pre-treated with mRPMI were harvested and subjected to gelatin zymography. The gels were scanned and the images were analyzed using imageJ. The relative secretion of MMP-9 was calculated from 3 experiments (*p < 0.05). e. Total cell lysate from serum-starved and mRPMI treated SK-N-SH cells were subjected to western blot analysis, and protein detection was carried out using anti-integrin α3 or anti-integrin β1 antibodies. Anti-actin antibody was used for loading control.

Fig 7. Effect of CXCL12 and mRPMI on invasion in neuroblastoma cells.

a. Neuroblastoma cells serum-starved (control) and pre-treated with CXCL12, were assayed for invasive potential using transwell invasion assays. Plotted is the mean number of cells migrating across the mesh from 5 experiments. b. Invasion index of neuroblastoma cell lines serum-starved (control) and pretreated with CXCL12. Plotted is the invasion index calculated from 5 experiments. c. Neuroblastoma cells serum-starved (control) and pre-treated with mRPMI, were assayed for invasive potential using transwell invasion assays. Plotted is the mean number of cells migrating across the mesh from 5 experiments. (*p<0.05) d. Invasion index of neuroblastoma cell lines serum-starved (control) and pretreated with mRPMI. Plotted is the invasion index calculated from 5 experiments. (*p<0.05)

Fig 8. Effect of mRPMI on CXCR4 ubiquitination.

CXCR4 immune precipitates from serum-starved (control) and mRPMI treated SK-N-SH cells were subjected to western blot analysis. Protein detection was carried out using anti-ubiquitin antibody. Shown is the representative result from 3 experiments.