Extracellular Vesicle-Based Method for Detecting MYCN Amplification Status of Pediatric Neuroblastoma

Figure 1. Characterization of two EV subtypes released from MYCN-amp and MYCN-NA NB cell lines. (a) Western blot analysis of the positive (Alix, Tsg101, Hsp70) and negative (calnexin) EV markers, and MYCN encoding n-Myc protein in cells, MVs and Exo of SK-N-BE2 and SH-SY5Y. The full-length blot images were provided in Figure S1. (b) Transmission electron microscopy of MVs and Exo released from MYCN-amp and MYCN-NA NB cells. The arrows indicate the nanoscale round-shaped vesicles with the size difference between MVs and Exo. Scale bar, 100 nm. (c) Nanoparticle tracking analysis showed the vesicle size distribution of MVs and Exo. (d) Quantitative RT-PCR revealed that MYCN mRNA could be detected in MVs, but not Exo, of MYCN-amp NB. The PCR products are shown on the left panel, while the MYCN mRNA expression (normalized to GAPDH) is demonstrated on the right panel. Abbreviations: 5Y, SH-SY5Y; BE2, SK-N-BE2; Exo, exosomes; MV, microvesicles; MYCN-amp, MYCN amplification; MYCN-NA, MYCN non-amplification.

Figure 2. Detection of MYCN amplification status of six NB cell lines and their MVs. (a) Fluorescent in-situ hybridization (FISH) using the DNA probes for MYCN (2p24; green) and CEP2 (2p11.1-q11.1; orange). Quantitative RT-PCR was performed to detect MYCN mRNA in (b) six neuroblastoma cell lines and (c) their corresponding MV isolates. The PCR products were validated on agarose gel electrophoresis. MYCN mRNA expression level was normalized to GAPDH. Abbreviations: 5Y, SH-SY5Y; AS, SK-N-AS; BE2, SK-N-BE2; DZ, SK-N-DZ; IMR, IMR-32; MV, microvesicles; MYCN-amp, MYCN amplification; MYCN-NA, MYCN non-amplification; SH, SK-N-SH.

Figure 3. MYCN-MV detection in the simulated serum specimens. (a) A schematic diagram represents the workflow of simulated sample generation by pulsing MVs derived from NB cells into the human serum. Three simulated serum samples were generated independently as per the NB cell lines. Then, the simulated serum samples were subjected to MV isolation, RNA extraction and MYCN mRNA measurement by qRT-PCR. (b) MYCN-MV detection in six simulated samples by qRT-PCR demonstrated by the agarose gel electrophoresis and the normalized MYCN mRNA expression to GAPDH. Abbreviations: 5Y, SH-SY5Y; AS, SK-N-AS; BE2, SK-N-BE2; DZ, SK-N-DZ; IMR, IMR-32; MV, microvesicle; MYCN-amp, MYCN amplification; MYCN-MV, MYCN mRNA-containing microvesicle; MYCN-NA, MYCN non-amplification; SH, SK-N-SH.

Figure 4. MYCN-MV detection in patient-derived bone marrow plasma specimens. MVs were isolated from 2 mL bone marrow plasma of NB patients and then were subjected to RNA extraction and qRT-PCR. The results are shown by the agarose gel electrophoresis (left panel) and the normalized MYCN mRNA expression to GAPDH (right panel). Abbreviations: BM, bone marrow; Dx, 1st diagnosis; HSCT, hematopoietic stem cell transplant; MYCN-amp, MYCN amplification; MYCN-MV, MYCN mRNA-containing microvesicle; MYCN-NA, MYCN non-amplification; Tx, ongoing treatment.