We did read with great interest the recent review published by Ichim et al on the
potential role of tumor exosomes as immune escape mechanism [1], and we were pleased
to see that the authors shared our original idea that these organelles may represent
a crucial tool of immunosuppression in cancer [2,3]. Indeed, although tumor cells
are well acknowledged to affect immune functions through the release of diverse soluble
factors or cell-to-cell contact mediated mechanisms [4,5], the involvement of alternative
pathways based on the secretion of membrane microvesicles has been so far largely
unappreciated [6]. Exosomes are endosome-derived organelles of 50–100 nm size, actively
secreted by virtually all cell types through an exocytosis pathway that is used under
normal as well as pathological conditions [6]. Their first description can be attributed
to the biochemist Rose Johnstone, who reported in her 1980s investigations about these
lipid-encased particles produced as a mechanism for shedding of specific membrane
functions during reticulocyte maturation [7]. Since then, these curious microvesicles
lingered in obscurity, although several reports kept referring to exosomes as potential
pathway utilized by different cell types to eliminate cellular material or establish
intercellular cross-talk [8]. Finally in 1996 these microparticles were recognized
for their central role in antigen presentation with the work of Graça Raposo and Hans
Geuze of Utrecht University in the Netherlands, who reported that exosomes secreted
by B cells could promote T cell cross-priming through the expression of HLA/peptide
complexes [6]. Based on these and following observations about the role of exosomes
in antigen presentation, the exacerbated production of these vesicles by tumor cells
was initially welcomed as a process potentially involved in the induction and maintenance
of tumor immunity [9]. Indeed, the expression of a large panel of tumor proteins with
antigenic properties, like MelanA/Mart-1 and gp100 in melanoma-derived exosomes, and
CEA and HER2 in exosomes produced by carcinoma cells [9-11], supported the role of
these organelles as cell-free source of tumor antigens for T cell priming and paved
the way to clinical trials based on vaccination with tumor exosomes in patients with
advanced disease [12].
However, following studies from several groups including ours have progressively suggested
that these vesicles, being close replicas of the originating cancer cells, could transport
not only antigenic material but also molecules responsible for the detrimental effects
exerted by tumor cells on the immune system [6,13,14].
As most researchers, we entered the exosome field by chance, in the course of studies
on FasL as tumor immune escape mechanism in human cancer. Indeed, despite the first
report on the expression of FasL by melanoma [15], we could not succeed in detecting
stable membrane expression of this pro-apoptotic molecule on such tumor cells. However,
by using immunocytochemistry and immunoelectron microscopy, we found that FasL was
indeed detectable intracellularly, as localized in defined endocytic compartments
with a clear secretory behaviour. Thanks to this initial observation, we discovered
that human melanoma as well as colon carcinoma cells constitutively release FasL and
TRAIL-expressing exosomes, which induce death by apoptosis in activated T cells [10,11].
This evidence, confirmed also by Whiteside and coworkers in head and neck cancer [16],
highlights a germane role of microvesicular structures in counteracting tumor immunity
by simply eliminating activated T cells bearing tumor-reactive TCR. This might occur
even at distance (in peripheral lymphoid organs, bone marrow, peripheral blood, and
biological fluids) without the need for a direct cell-to-cell contact. And given the
evidence that exosome of probable tumor origin are abundantly found in plasma or pathological
effusions of cancer patients [9,11], it can be easily hypothesized that this pathway
may contribute to the in vivo moulding of immune as well as other cancer-related host
responses. More recent studies have then reported that the detrimental effect of tumor
exosome on immune effector functions is not restricted to T cells but can target NK
cells as well, through the skewing of IL-2 responsiveness in favour of regulatory
T cells [17] or down-modulation of NKG2D expression [18]. Moreover, the negative influence
of tumor exosomes on specific immunity goes beyond T and NK cells and may also target
crucial up-stream steps for T cell cross-priming, namely dendritic cell (DC) differentiation.
In fact, we have more recently observed that the presence of tumor exosomes during
monocyte differentiation into DC skews the whole process toward the generation of
aberrant cells expressing myeloid markers (such as CD14 and CD11b), lacking or bearing
low levels of co-stimulatory molecules (like HLA-DR, CD80 and CD86) and spontaneously
secreting TGF-beta [19,20]. These cells, which exert a strong immunosuppressive activity
on T cell proliferation and function, highly resemble the "myeloid-derived suppressor
cell" subset described to accumulate with tumor progression in different murine models
[21]. Interestingly enough, melanoma patients with advanced disease have high levels
of these CD14+ HLA-DR neg/low TGF beta-secreting cells in their peripheral blood,
and this frequency appears to be a disadvantageous factor for the development of immune
responses to tumor vaccines [20]. These findings, which again were confirmed in other
experimental settings [22], define a very sharp profile of tumor exosomes as efficient
delivery system of immunosuppression, contributing to the maintenance of an immune
tolerance state in cancer bearing hosts.
The interest on exosomes has recently spread out as these vesicles are being found
involved in a wide spectrum of physiological and pathological cellular events, as
alternative tools of intercellular communication and paracrine functions [23], or
as pathogenic pathways in viral [24] and prion-related diseases [25]. Thanks to their
peculiar lipid composition, highly enriched in ceramide [26], sphingomyelin, cholesterol
and GM3 glycolipid [27], exosomes may serve as a more advantageous carrier of signal
delivery favouring stable conformational conditions, increased bioactivity, improved
bio-distribution and amplified target interaction of their protein content with respect
to soluble molecules. In the last years, literature is indeed flourishing with examples
proving the role of tumor exosomes in the transfer of growth factors and cognate receptors
to homologous or heterologous target cells. For instance glioma cells can share EGFR
by intercellular transfer of membrane-derived microvesicles ('oncosomes') [28], or
pancreatic carcinoma can deliver exosomes overexpressing tetraspanin family members
and promoting autocrine secretion of MMP and VEGF [29]. The evidence that these organelles
can also shape protein synthesis through the transfer of functional mRNAs and microRNAs,
as recently reported in transformed mastocytes [30], adds then a further pathway to
the potential modulating properties of these peculiar organelles.
If tumor exosomes are such a powerful instrument of environmental shaping, then getting
rid of them should significantly affect cancer cell ability to survive and expand
in vivo. In their review, Ichim et al propose a physical approach based on the extracorporeal
removal of exosomes from plasma of cancer patients, through a novel hollow-fiber cartridge
(Hemopurifier™) designed to eliminate particles expressing heavily glycosylated surface
proteins, like in case of viruses and cancer microvesicles [1]. The approach could
be further implemented by the attachment of clinical grade molecules and antibodies
to the cartridge resin, to allow microvesicle depletion on the basis of selected marker
expression. Although interesting, feasible and potentially effective in the short-term,
this strategy could only have an impact on circulating exosomes, leaving vesicles
accumulating at tumor tissue level, in draining lymph nodes or in other relevant lymphoid
compartments, still available for immunosuppressive functions. Obviously, physical
removal would not interfere with the process of exosome secretion, and would indiscriminately
eliminate vesicles from both pathological and normal cells. In alternative, we are
considering to intervene on tumor exosome secretion by inhibiting up-stream crucial
pathways involved in the process. Although definitive information on the mechanisms
regulating microvesicle release by cancer cells are presently scantly, preliminary
data suggest that particular molecules, such as drugs interfering with microtubule
stability (taxanes and vinca alkaloids) [M. Iero, unpublished observations] or additional
microtubule-disturbing molecules like vincristine [31], can affect endosomal stability
and reduce microvesicle release. Similarly, drugs targeting the activity of enzymatic
efflux pumps expressed on acidic vacuoles, such as vacuolar-ATPases inhibitors, could
selectively alter exosome trafficking and release in tumor cells [Iero et al., unpublished,
[32]]. Benefits from modulation of exosome secretion could also come from qualitatively
shaping protein composition of secreted microvesicles with drugs altering biological
features of tumor vesicles, such in the case of curcumin, a natural polyphenol which
has been shown to reduce immunosuppressive functions of breast carcinoma-secreted
exosomes [33].
A more specific approach would be instead to identify the molecular mechanisms responsible
for the immunosuppressive activity and the microenvironment remodelling effects of
tumor exosomes [34], to selectively interfere with these pathways through specific
antibodies, antisense oligonucleotides or signalling inhibitors.
Independently from the tool utilized for diminishing exosome release by tumor cells,
the most challenging task of the near future is to prove that interfering with microvesicle
secretion in vivo may indeed result in tumor growth arrest or slow-down thanks to
the recovery of specific immunity and the interruption of paracrine/autocrine loops
in tumor microenvironment. Prior to any clinical intervention, experimental studies
in animal models should thus be performed to assess what is the real impact that these
vesicles play in cancer progression and what is the expected benefit of shutting off
their production at tumor site.
Authors contributions
VH was responsible for editorial writing, senior scientist responsible for the studies
on the immunosuppressive functions of tumor exosomes. PF was responsible for editorial
reviewing, scientist responsible for the studies on the induction of myeloid-derived
suppressor cells by tumor exosomes. MI was responsible for editorial reviewing,
scientist responsible for the studies on the modulation of exosome release by tumor
cells. SF was responsible for editorial reviewing, external collaborator in the
studies on the involvement of proton-pump inhibitors on exosome release. LR was
responsible for editorial writing and reviewing, supervisor of the studies on tumor
exosomes