Exosomes in melanoma: a role in tumor progression, metastasis and impaired immune system activity

Abstract

Exosomes (Exo) are small vesicles produced by melanoma cells and the accessory cells of the tumor microenvironment. They emerge via both classical and direct pathways and actively participate in tumor colonisation of distant tissues. The proteins, nucleic acids, cytokines and growth factors engulfed by Exo are transferred to recipient cells, where they drive numerous functions required for the tumor escape from immune system control and tumor progression. By positively or negatively modulating immune cell properties, Exo provoke immune suppression and, in turn, defective dendritic cell (DC) functions. Together, these effects limit the cytotoxicity of T-cells and expand both T-regulatory and myeloid-derived suppressor populations. They also hinder perforin and granzyme production by natural killer cells. Finally, Exo also control the organotropism of melanoma cells. The distinct phenotypic properties of Exo can be exploited both for diagnostic purposes and in the early identification of melanoma patients likely to respond to immunotherapy. The potential therapeutic application of Exo derived from DCs has been demonstrated in vaccination trials, which showed an increase in anti-melanoma activity with respect to circulating tumor cells. However, additional studies are required before Exo can be effectively used in diagnostic and therapeutic applications in melanoma.

Keywords: exosomes; immune system; melanoma.

Figure 1. The pathways of exosome formation

Two major pathways of Exo formation have been described as ‘direct’ and ‘classical’ (left). The ‘direct pathway’ promotes the Exo formation through the direct exocytosis of vesicles, including nanovesicles and MV stemmed from the outward budding of the plasma membrane. Contrariwise, the ‘classical pathway’ requires the re-activation of endosomes that originates from the inward budding of plasma membrane and leads to the development of multi-vesicular endosomes (MVE). After the active packaging of their content, MVE fuse with the plasma membrane and release Exo in the extracellular space. Exosomes consist of a lipid bilayer (right) that contains trans-membrane and cytoplasmic proteins or non-coding miRNAs, mRNA as well as single-stranded and double-stranded DNAs. Also, exosomes are engulfed of a number of proteins that are mostly represented by tumor-derived neo-antigens, class-I and -II MHC molecules and tetraspanins. Furthermore, the early phases of melanomagenesis are characterized by the production of melanosomes, melanin-containing organelles that result highly enriched of oncogenic miRNAs, whose major role concerns the preparation of the primary metastatic niche milieu.

Figure 2. Exosomes drive the metastasis of melanoma cells

Primary melanoma cells produce Exo that are able to target distinct populations, both within the tumor microenvironment (left) and at distant sites where they participate to the pre-metastatic niche formation (right). The molecular mechanisms by which Exo interact with target cells include the cytokines, miRNAs and receptors variably expressed by or accumulated in these vesicles. Exo enriched in Let7a, Let7i, miR-191 and miR-222 (A) promote tumor growth and invasiveness while are also able to impair immune system activity, leading to the defective maturation of dendritic cells, impaired cytotoxicity of T-cells and the expansion of suppressive populations, including regulatory T-cells (Treg) and myeloid-derived suppressor cells (MDSCs). (B) The expression by Exo of miR-9 and the high levels of both interleukin (IL)-6 and vascular endothelial growth factor (VEGF) drive the neo-angiogenesis, through the activation of the JAK/STAT pathway, whereas uPAR and ADAMs proteases (C) promote the remodelling and degradation of the extracellular matrix. Melanocytes produce melanosomes (D) as vesicles engulfed of melanin that are found accumulated in keratinocytes. Malignant melanoma cells stimulate the collagen-associated fibroblasts (CAFs) of the dermis leading to increased melanoma cell proliferation through the over-production of miR-149, -211, -23, -let7a and -let7b. In addition, the up-regulation of CD169 by Exo is a key step in the recruitment of melanoma cells to lymph nodes (E), whose colonisation by metastatic cells is also favoured by α_v integrins, hypoxia-inducible factor (HIF)-1α and tumor necrosis factor (TNF)-α. In addition, specific organotropism (F) is driven by MET-expressing Exo, which in turn promotes the mobilisation of bone marrow progenitors (BMDCs) implicated in neo-vasculogenesis and pre-metastatic niche formation.

Figure 3. Exosomes from melanoma cells balance immune system activity

A functional anti-melanoma immune response is orchestrated by Exo containing inhibitory and stimulatory molecules that may alter the immune system balance. The expansion of Tregs and MDSCs is also induced by up-regulated transforming growth factor (TGF)-β or by inhibitory signals directed from melanoma-derived Exo toward both CD8⁺ T-cells and DCs. Among the immune-suppressive cytokines involved in these processes are interleukin (IL)-6 and IL-10 as well as pro-apoptotic molecules, all of which may be transferred from Exo to immune cells. Conversely, exosomal TGF-β and toll-like receptor (TLR) activation impair the release of perforins and granzyme by NK cells as well as macrophage activation. However, also immune cells produce Exo within the microenvironment and those from DCs (D-Exo) and CD8⁺ T-cells (T-Exo) interplay with both tumor cells and immune cells leading to melanoma evasion from the immune system control.