BACKGROUND: Microenvironment cues involved in melanoma progression are largely 
unknown. Melanoma is highly influenced in its aggressive phenotype by the 
changes it determinates in its microenvironment, such as pH decrease, in turn 
influencing cancer cell invasiveness, progression and tissue remodelling through 
an abundant secretion of exosomes, dictating cancer strategy to the whole host. 
A role of exosomes in driving melanoma progression under microenvironmental 
acidity was never described.
METHODS: We studied four differently staged human melanoma lines, reflecting 
melanoma progression, under microenvironmental acidic pHs pressure ranging 
between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage 
and environmental pH, we applied a technique to generate native fluorescent 
exosomes characterized by vesicles integrity, size, density, markers expression, 
and quantifiable by direct FACS analysis. Functional roles of exosomes were 
tested in migration and invasion tests. Then we performed a comparative 
proteomic analysis of acid versus control exosomes to elucidate a specific 
signature involved in melanoma progression.
RESULTS: We found that metastatic melanoma secretes a higher exosome amount than 
primary melanoma, and that acidic pH increases exosome secretion when melanoma 
is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to 
show that acidic pH influences the intercellular cross-talk mediated by 
exosomes. In fact when exposed to exosomes produced in an acidic medium, pH 
naïve melanoma cells acquire migratory and invasive capacities likely due to 
transfer of metastatic exosomal proteins, favoring cell motility and 
angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in 
acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, 
HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor 
prognosis, whose high expression was in part confirmed in bioptic samples of 
lymph node metastases.
CONCLUSIONS: A crucial step of melanoma progression does occur at melanoma 
intermediate -stage, when extracellular acidic pH induces an abundant release 
and intra-tumoral uptake of exosomes. Such exosomes are endowed with 
pro-invasive molecules of clinical relevance, which may provide a signature of 
melanoma advancement.