Assessment of MART-1 and HMB-45 in plasma-derived extracellular vesicles from patients with melanocytic lesions

Melanoma is a poor prognosis cancer type originated in the cutaneous tissue that releases extracellular vesicles (EVs) to prepare the metastatic niche. In addition, EVs are important mediators in proinflammatory states. However, it is not clear if plasma EVs, derived from melanocytes (MART-1 or HMB45), may be used as prognostic markers for melanoma. Thus, we proposed to detect MART-1 and HMB-45 in plasma-derived EVs from patients with melanoma and nevi, submitted to surgical resection attended at the Dermatology Division of the Hospital Universitário Antônio Pedro/ESBERH (CAAE:64852022.1.0000.5243). EVs were isolated by a differential centrifugation (12.000xg 2 minutes 4ºC and 20.000xg 20 minutes 4ºC) and characterized by nanoscale flow cytometry (nFC, CytoFLEX S). Antibody concentrations to determine melanocytic EVs were: annexin V-FITC (Biolegend) 0.36 ug/mL, anti-Human MART-1 Alexa Fluor 647 (BD Biosciences) 0.8 ug/mL and anti-Human HMB-45 Alexa Fluor 790 (Santa Cruz) 1.2 ug/mL. We also performed Nanoparticle Tracking Analysis (NTA) to confirm the existence of particles. Twelve participants, 83.3% of females (n=10) and with a mean age of 68.4±12.9 years were included. Of these, six (50%) had melanoma. In NTA, we observed that EVs from the nevi group were slightly bigger (nm) compared to the melanoma group (85.9±5.3 vs. 80.1±2.9 nm). Also, a lower count of particles was observed in melanoma patients (6.45x10¹⁰±3.11x10¹⁰ vs. 1.89x10¹¹±9.19x10¹⁰ particles/mL), which was confirmed by nFC. Lastly, we identified that melanoma patients presented higher counts of HMB-45 positive EVs (1.05x10⁵±1.71x10⁵ vs. 9.46x10⁴±9.59x10⁴ counts/mL) but not MART-1 positive EVs (1.25x10⁵±9.85x10⁴ vs. 2.15x10⁵±1.83x10⁵ counts/mL). Therefore, our data suggest that melanocyte-derived EVs are present in plasma and melanoma patients present more HMB-45 positive EVs. However, the plasma EVs from this group were slightly smaller, which may be related with its cargo.