Exosomes from N-Myc amplified neuroblastoma cells induce migration and confer chemoresistance to non-N-Myc amplified cells: implications of intra-tumour heterogeneity

Abstract

Neuroblastoma accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressiveness of the disease is poorly understood. Exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. Hence, characterising the exosomal protein components from N-Myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. In this study, a comparative proteomic analysis of exosomes isolated from cells with varying N-Myc amplification status was performed. Label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc amplified SK-N-BE2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from N-Myc knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. These findings suggest that exosomes could play a pivotal role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. Abbreviations: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; FCS = foetal calf serum; NTA = nanoparticle tracking analysis; LC-MS = liquid chromatography-mass spectrometry; KD = knockdown; LTQ = linear trap quadropole; TEM = transmission electron microscopy.

Keywords: N-Myc; Neuroblastoma; chemoresistance; exosomes; intra-tumour heterogeneity.

Figure 1.

Characterization of exosomes isolated from N-Myc amplified and non-amplified neuroblastoma cells. (a) Western blot analysis of exosomal enriched proteins Alix and TSG101 in fractions obtained from OptiPrep density gradient centrifugation (SK-N-BE2 cells). TSG101 and Alix were enriched in fractions 7 and 8 corresponding to the buoyant density of 1.13 and 1.15 g/mL. (b) Western blot analysis representing the presence of Alix and TSG101 that are enriched in exosomes derived from SH-SY5Y cells. Fraction 7 contained a high abundance of Alix and TSG101. (c) TEM images of exosomes isolated by OptiPrep density gradient centrifugation suggested the presence of vesicles. (D) Venn diagram representing proteins present in exosomes derived from N-Myc amplified (SK-N-BE2) and non-amplified (SH-SY5Y) neuroblastoma cells. A total of 749 proteins are found to be common between the exosomes isolated from the two neuroblastoma cell lines. (e) Venn diagram depicting differentially abundant (>2-fold) proteins in SK-N-BE2 and SH-SY5Y cell-derived exosomes. A total of 581 proteins were enriched in exosomes derived from SK-N-BE2 cells compared to the exosomes from SH-SY5Y cells. Similarly, a total of 385 proteins were enriched in exosomes isolated from SH-SY5Y cells. The red arrow represents proteins that are of high abundance in SK-N-BE2 cell-derived exosomes compared SH-SY5Y cell-derived exosomes. Green arrow represents proteins that are of lower abundance in SK-N-BE2 cell-derived exosomes compared SH-SY5Y cell-derived exosomes.

Figure 2.

Proteomic analysis of SK-N-BE2 cell-derived exosomes are enriched with exosomal and signalling proteins. (a) Heatmap of exosomal enriched proteins identified in exosomes isolated from neuroblastoma cells. Alix (PDCD6IP) was highly enriched in both the exosomal samples. (b) RAB proteins were differentially abundant in exosomes isolated from SK-N-BE2 and SH-SY5Y cells. RAB10, RAB14, RAB1B, RAB5C and RAB7A were detected in both the exosomal samples in high abundance. (c) The abundance of integrins in exosomes isolated from SK-N-BE2 and SH-SY5Y cells. (d) Western blotting based validation of proteins found in exosomes secreted from N-Myc amplified and non-amplified neuroblastoma cells. WCL = whole cell lysates. (e) FunRich based enrichment analysis of signalling pathways enriched in proteins differentially abundant in SK-N-BE2 and SH-SY5Y cell-derived exosomes. * denotes p < 0.05, ** denotes p < 0.01 and *** denotes p < 0.001 as determined by hypergeometric test.

Figure 3.

Exosomes derived from SK-N-BE2 cells induce migration in SH-SY5Y cells. (a) Colony forming abilities of SH-SY5Y cells treated with and without exosomes. SH-SY5Y cells (200 cells per well) were incubated with 10 µg/mL of exosomes isolated from SK-N-BE2 and SH-SY5Y cells. The SH-SY5Y cells in the presence of SK-N-BE2 cell-derived exosomes showed a significant increase in colony forming ability (n = 3). (b) Transwell migration assay performed on SH-SY5Y cells after treating with exosomes (10 µg/mL) isolated from SK-N-BE2 and SH-SY5Y cells. There was a significant increase in the migratory ability of SH-SY5Y cells upon SK-N-BE2 cell-derived exosomes treatment (n = 3). (c) Wound healing assay was performed on SH-SY5Y cells (n = 3). The monolayer of cells closed the wound faster when the cells were treated with SK-N-BE2 cell-derived exosomes (10 µg/mL). Error bars represent the standard error of mean, * denotes p < 0.05 and ** denotes p < 0.01 as determined by student’s t-test.

Figure 4.

Exosomes derived from SK-N-BE2 cells conferred chemoresistance to SH-SY5Y cells. FACS apoptosis assay was performed on SH-SY5Y cells after treatment with SK-N-BE2, SH-SY5Y and N-Myc KD cell-derived exosomes (5, 10 and 20 μg/mL), doxorubicin (1 µM) and combinational treatment of exosomes and doxorubicin. As shown in the graph, SH-SY-5Y cells obtained resistance to doxorubicin in the presence of SK-N-BE2 cell-derived exosomes (n = 3). Error bars represent the standard error of mean, * denotes p < 0.05, ** as determined by student’s t-test.