Potential role of exosome-associated microRNA panels and in vivo environment to predict drug resistance for patients with multiple myeloma

Abstract

Multiple myeloma (MM) is the second most common hematologic neoplasms and an appropriate in vivo environment for myeloma cells has potential implications for initiation, progression, and metastasis of MM. Exosomes, entities carrying microRNAs (miRNAs) to target locations, participate in the cross-talk between myeloma cells and nonmalignant components of the in vivo environment. This study disclosed the emerging roles of circulating exosome-associated miRNAs in drug resistance (DR) of MM. To this end, the medical records of consecutively hospitalized MM patients, who received novel agents-based therapies, were analyzed. Then, an optimized procedure was established for exosome isolation and exosomal RNA analysis. The exosome-associated miRNA expression patterns for predicting bortezomib (Bz) resistance of MM were further examined using a microarray. In total, 204 patients were enrolled with DR rates of 36.5%, 73.1% and 81.8% in the bortezomib (Bz), thalidomide and lenalidomide containing groups. The serum total light chain ratio ≥ 100, CRP ≥ 20 mg/L, and the second-line usage increased risks of acquired Bz-resistance. Among 68 cases having genetic tests, a high risk factor for predicting de novo DR was 1q21 amplification, which also correlated with lower levels of cholesterol and LDL-C. Moreover, nano-sized exosomes were isolated with significantly increasing internal RNAs and down-regulation of exosomal miR-16-5p, miR-15a-5p and miR-20a-5p, miR-17-5p was revealed in the patients resistant to Bz. The routine workup of MM hardly suggested a value for DR prediction. The circulating exosomes carrying miRNAs provided a window that permits a better understanding of the in vivo intercellular crosstalk in MM patients.

Keywords: exosome; microRNA; microvesicle; myeloma; resistance.

Figure 1. Isolation and validaton of exosomes from both U266 cell line and MM patients

MM-derived exosomes were isolated and purified as depicted in section A. In section B, presence of MVs and exosomes: Both MVs and exosomes were present by TEM (A) and Nanosight (B) following centrifugation of U266 culture supernatant without removal of MVs by 16 000 g × 60 min; Exosomes were present by TEM (C) and Nanosight (D) following centrifugation of U266 culture supernatant with removal of MVs by 16 000 g × 60 min. Processed plasma samples from MM patients based on the protocol in section A, exosomes derived from MM quantitatively predominated (F) than those from the healthy control (E). In section C, EMVs, exosomes and MVs were isolated from the culture supernatant of U266 cells, respectively. Exosomal marker proteins of HSP70 were detected for both exosomes and MVs, while CD63 was mainly observed in exosomes.

Figure 2. The differential expression of exosome-associated miRNAs in the subgroups of MM

Exosomal RNAs were detected from both the culture supernatant of U266 cells and the plasma samples from MM patients (A). Significant differences (* < 0.05) of exosomal RNA content were observed between the Bz-response group (E) and Bz-resistant group (G). Exosomal RNA content of both groups was much higher than the content of circulating RNAs (D and F) extracted using the miRNeasy Serum/Plasma Kit (Qiagen). In sections (B and C) compared to the Bz-response group, 83 miRNAs were expressed at higher levels and 88 miRNAs were expressed at lower levels in the Bz-resistant group among 3180 miRNAs on the microarray. In section D, a miRNA-RNA synergistic network was constructed with miR-17-5p, miR-20a-5p and miR-15a-5p, miR-16-5p exhibiting more synergism in the Bz-resistant mechanism of MM.