Our review follows the “Minimal information for studies of extracellular vesicles 2018 (MISEV2018)” updated guidelines [1
]. In order to guarantee correct divulgation and to increase the experiment reproducibility, vesicles nomenclature, characteristics and cited papers have been selected to follow the recommendations from the International Society for Extracellular Vesicles (ISEV) as closely as possible.
Small extracellular vesicles (small EVs), sometimes widely called exosomes, are vesicles (30–150 nm) released by all cells present in all body fluids that are involved in short- and long-distance cell communication [2
]. Small EVs are typically included in the category of extracellular vesicles (EVs), together with exosome-like vesicles (20–50 nm), membrane particles (50–80 nm), microvesicles (100–1000 nm) and apoptotic bodies (<5000 nm) [1
]. Beside the size, what distinguishes small EVs from other extracellular vesicles is their specific biogenesis, which is strictly bound to the endosomal compartment [1
The release of small EVs is dependent on the cell of origin, the physiological context, and the purpose [8
]. Initially considered a physiological phenomenon to remove cellular waste, today, small EVs are considered to be a sophisticated way for cells to communicate with the surrounding microenvironment [7
]. Their molecular content is strictly dependent on the cell of origin [10
]. Interestingly, it was shown that tumor-derived small EVs (TEVs) carry a cargo of molecules that is different from the small EVs released by the healthy counterpart [10
]. Therefore, it is not surprising that tumor cells release specific small EVs containing an unique molecular fingerprint which is used by tumors to increase proliferation [12
], metastasis formation [12
] and immune escape [14
], effectively reshaping the healthy microenvironment in favor of a pro-tumorigenic one.
3. Hematological Malignancy-Derived Small EVs and Immune Cells
Of the wide ranging of studies describing the role of TEVs in tumorigenesis, in this review, we will focus our attention on hematological malignancy-derived small EVs and their impact on immune and stromal cells of the tumor microenvironment (TME).
The ability to evade the immune surveillance is one of the strategies for the generation of a proper tumor niche and a successful tumor development [80
]. This process is not only visible in the area of the primary tumor but it is also a way to generate pre-metastatic and metastatic niches [80
]. One quality of the TEVs is to carry molecules which are used in several level of communications with the surrounding cells of the microenvironment. Through receptor-mediated uptake, TEVs release their content into the cytoplasm by directly fusing with the cell membrane. Furthermore, TEV-carried ligands are recognized by proper receptors on target cells; for example, antigens carried by TEVs can bind to major histocompatibility complex (MHC) receptors [82
]. Altogether, it is believed that small EVs play an important role in the communication of tumor cells to non-malignant bystander cells in their surroundings, in order to create a tumor-friendly environment (Figure 1
3.1. B Cells
B cells are an essential component of the immune system. They are mainly responsible for modulating immune response and inflammation through the production of antibodies and the promotion of T cell activation and proliferation through antigen presentation [83
]. Recent studies have revealed a new category of B cells, known as regulatory B cells (Bregs), involved in the control of anti-tumor immune response and tumor development [84
]. Bregs possess protective functions, maintain immune tolerance and suppress pathological autoimmune and inflammatory responses [89
]. Despite that, Bregs have been shown to play an important role in supporting cancer immune escape through the release of anti-inflammatory mediators, such as interleukin-10 (IL-10) [91
]. Release of IL-10 allows Bregs to regulate CD4+
T cell differentiation, proliferation and activity (cytokine secretion), for instance by pro-apoptotic signals [90
]. In a similar way, Bregs suppress CD8+
T cell immune response and function [94
], natural killer IFN-γ production [97
], monocyte proliferation and cytokine release [98
] and finally dendritic cell IL-12 production [99
]. Furthermore, the release of IL-10 promotes the expansion of Bregs which cause an enhanced immune depression together with IL-10 dependent stimulation and expansion of Tregs [97
Bregs are playing an important role in lymphoma. IL-10 is rapidly produced and released by Bregs stimulated with lymphoma-derived small EVs [102
]. Furthermore, small EVs released by Burkitt’s lymphoma cell lines stimulate B cell proliferation, induction of activation-induced cytidine deaminase (AID), and the production by B cells of circle and germline transcripts for IgG1 [103
Haque and Vaiselbuh suggested that acute lymphoblastic leukemia (ALL)-derived small EVs regulate in vitro leukemic and non-leukemic B cell proliferation through the transport of proliferative, pro-survival and anti-apoptotic factors [104
]. Finally, Patel et al. reported that primary human ALL-derived small EVs are released by the growing leukemia clones to promote proliferation and survival of the low density growing clones [105
Despite having canonical protective activities, B cells and Bregs, targeted by TEVs, can be used by the tumor to circumvent the immune system and enhance tumor growth.
3.2. T Cells
Interaction between TEVs and T cells takes place through the interaction with surface molecules which generate signals resulting in sustained Ca2+
flux and activation of downstream signaling pathways, leading to alterations in the T cell transcriptome [82
Cancer cells use TEV potential in order to diminish T cell function and thus weaken the overall immune response. Whiteside et al. reported that TEVs mediate the inhibition of CD3ζ chain expression and drastically reduce its mRNA levels [106
]. Decreased expression of the T-cell receptor (TCR) ζ-chain has been reported in several autoimmune, inflammatory and malignant diseases (e.g., lymphoma), and it is commonly associated with suppression of T cell proliferation and altered cytokine production [107
]. During cancer progression, T cells are found in close contact with tumor cells and represent an essential component of the TME. In line with this, it was shown that subverted CD4+
T cell subsets within the tumor microenvironment may exhibit a tumor-promoting activity [111
Hematopoietic malignancies actively use small EVs to strike on T cells and cause a wide range of effects meant to reduce T cell actions on tumor development. Smallwood et al. demonstrated that autologous patient CD4+
T cells internalize chronic lymphocytic leukemia (CLL)-derived small EVs containing miR-363 that targets the immunomodulatory receptor CD69, which leads to inhibit the migration of effector T cells [112
]. In another study, Diffuse Large B Cells Lymphoma (DLBCL)-derived small EVs were shown to be rapidly captured by T cells, leading to either PD-1 up-regulation or to pro-apoptotic signals, probably due to increased expression of Fas, FasL, and TRAIL [55
Chemotherapy is still widely used in cancer treatment. Specific TEVs are released by tumor cells under chemotherapy as a resistance mechanism to improve cancer cell survival and strongly reduce the immune response. In line with this, it was recently shown that B lymphoma-derived small EVs enriched with CD39 and CD73 are able to hydrolyse ATP released from chemotherapy-treated cancer cells into adenosine [116
] which is known to affect cancer immune response causing M2-like macrophages polarization and inhibit T cell activity and proliferation [117
]. Inhibition of T cell functions is essential in the process of immune escape. In this contest, TEVs are used to inhibit T cell activation and proliferation, as well as to increase pro-apoptotic signals.
3.3. Dendritic Cells
Dendritic cells (DCs) are widely distributed antigen-presenting cells (APCs) which have the unique capacity to induce the activation and differentiation of naive T lymphocytes [118
TEVs are used against DCs to inhibit their maturation and thus, the ability to activate effector cells. Lymphoma-derived small EVs carrying molecules such as TGF-β, IL-6 and prostaglandin E2 (PGE2) highly affect DC differentiation, maturation and function [120
]. Stimulated by TEVs, myeloid precursors, which usually give rise to DCs in the bone marrow, differentiate as well into myeloid-derived suppressor cells (MDSCs), a subtype of myeloid cells with pro-tumorigenic and immune suppressive properties [120
It has been shown that DCs efficiently capture DLBCL-derived small EVs, but this doesn’t cause apoptosis or upregulation of immunosuppressive mediators as it happens for T cells, but rather an enhancement of the tumor-specific immune response [122
]. In according with this, a recent study suggested a potential anti-myeloma vaccine strategy using a human myeloid leukemia cell line differentiated into DCs, known as DCOne vaccine [123
]. The potential of this cell line resides in its ability to produce a vast range of anti-MM antigens encapsulated in small EVs [125
]. When co-cultured with MM patient peripheral blood, DCOne vaccine-derived small EVs boost the expansion and activation of CD8+
T cells. Primary MM cells co-cultured with these DCOne vaccine-activated CD8+
T cells were efficiently lysed [123
The dual role of TEVs in modulating antitumor immunity is still poorly understood. Nevertheless, there is evidence for a possible modulation of the tumor immune response based on TEVs interaction with immune cells. For this reason, the attention is increasingly moving towards the use of more potent TEV-re-educated DCs to prevent, treat or eradicate tumors [126
3.4. Natural Killer Cells
Natural killer (NK) cells represent a further component of the innate immune system. These essential cytotoxic lymphocytes control microbial infections and tumor progression in a process regulated by a balance of activating and inhibitory signals [128
In cancer patients, a reduction of NK cell number as well as a depression of their activity have been previously correlated to the decrease expression of specific NK cell–activating receptors (NKp30, NKp46, NKG2C, and NKG2D) [74
]. TEVs are able to downregulate the expression and activity of NK cell–activating receptors, among which NKG2D is the most affected. Indeed, under thermal and oxidative stress, it has been described how T- and B-leukemia/lymphoma cells release small EVs enriched in NKG2D ligands which has been suggested to act as powerful decoy to downregulate NKG2D. [74
]. Beside this ligand-receptor interaction, also soluble growth factors released by tumor cells, and contained into TEVs, impair NK activity.
Sera of AML patients were shown to contain high levels of TEVs carrying CD33, CD34, CD117, MICA/MICB, and TGF-β1 which ultimately lead to immune suppressive effect due to decrease cytotoxic activity of NK cells. It is believed that the phosphorylation of SMAD and the down-regulation of NKG2D are the key processes to impair NK cell activity [74
]. In line with this, CML-derived small EVs have also been found to be typically enriched in TGF-β1, which has also been shown to be essential for the tumor cell proliferation [133
Furthermore, MM cells previously exposed to sub-lethal doses of the alkylating agent melphalan are capable of releasing small EVs stimulating the production of interferon-gamma (IFN-γ) by NK cells through a mechanism based on the activation of the nuclear factor-kappa B (NF-κB) pathway in a TLR2/heat shock protein 70 (HSP70)-dependent manner [134
]. In different circumstances, MM-derived small EVs also reduce cytotoxic activity of NK cells against MM cells [135
]. Furthermore, the ectoenzyme CD38, carried by MM-derived small EVs, has been suggested to convert nucleotides to adenosine, leading to an anergic immune system [136
Finally, despite being currently under further investigation, CD38 has also been suggested as possible player of MM-derived small EV internalization in immune cells such NK, monocytes, and myeloid-derived suppressor cells, possibly leading to an alternative strategy for tumor immune escape [136
]. NK cells are active players in the process of tumor cell disruption, thus TEVs are deployed with the aim to decrease NK cytotoxic activity and keep their number reduced.
Among the leukocytes, monocytes represent a subgroup of cells with a plasticity to differentiate into macrophages or dendritic cells. Monocyte plasticity is considerably reduced by TEVs. Indeed, lymphoma-derived small EVs efficiently interact with monocytes by membrane fusion, inducing secretion of the pro-inflammatory cytokines IL-6, TNF-α, IL-1β, and profoundly altering the process of their differentiation into dendritic cells [137
]. In line with the previously reported changes, we showed how CLL-derived small EVs containing the non-coding RNA hY4 were able to induce monocyte polarization, leading to cytokines release, such as CCL2, CCL4 and IL-6 and expression of PD-L1, suggesting a potential small EV-based mechanism of immune escape [138
Due to their possibility to differentiate into different immune cells, monocytes can be led to polarize into a pro-tumorigenic form, which ultimately decrease the pool of macrophages and DCs used in the fight against cancer development.
With a broad pro-inflammatory, destructive, scavenging and remodeling potential, macrophages are considered as key mediators of the immune response. Macrophages are highly plastic cells which constantly alter their functional state in response to environmental changes. The latter stimulate the expression of different surface markers and functional programs ultimately leading to the macrophage polarization [139
The phenomenon of polarization grants macrophages a double role in cancer. Canonical activated macrophages M1 are crucial for tumor immune response due to their ability to produce pro-inflammatory cytokines and reactive oxygen/nitrogen species. The M2 variation, on the other hand, produces anti-inflammatory cytokines which not only causes a considerable reduction of tumor immune response activity but also enhancing tumor progression through angiogenesis and promotion of matrix remodeling [139
In a cancer microenvironment, M2 macrophages are educated into Tumor-Associated Macrophages (TAMs). This category of macrophages is known to release pro-tumorigenic growth factors, chemokines and cytokines which will support tumor progression [140
Macrophages behavior can be regulated through TEVs. In a subcategory of DLBCL, the innate immune-signaling adaptor myeloid differentiation primary response 88 (MyD88) has been detected in the DLBCL-derived small EVs. Manček-Keber et al. described that MyD88 is transferred into macrophages triggering the activation of pro-inflammatory signals (such as NF-κB) independent from the TLR and IL-1R receptors [144
]. In another study, Chronic Myeloid Leukemia (CML)-derived small EVs have been shown to induce M2-like macrophage polarization leading to IL-10 and TNF-α overexpression. Furthermore, the downregulation of the inducible nitric oxide synthase (iNOS) causes reduction of nitric oxide (NO) and ROS levels in the TEV-treated macrophages [145
]. Similarly to the monocytes, macrophages are modified by TEVs and turn against the microenvironment. Together with the B cells, TAMs are used to reshape the microenvironment by enhancing tumor immune escape and promoting angiogenesis.
Neutrophils are the body’s first line of defense against foreign invaders and thus one of the most important key mediators of the innate immune response [146
]. Neutrophils are potent antitumor effector cells due to their cytotoxic activities and ability to release cytokines and chemokines which leads to the recruitment of other cells with antitumor activity [147
]. Despite this, similarly to macrophages and monocytes, neutrophils also show a phenotypic plasticity, which is modulated by different tumor-derived signals, and then display pro- or anti-tumor effects [150
CML can be driven by the formation of the hybrid gene BCR/ABL kinase as results of the Philadelphia chromosome rearrangements. CML-derived small EVs have been documented to carry such gene and transfer it in vitro and in vivo to neutrophils, ultimately causing an aberrant gene expression program in the target cells, and recapitulating CML-like symptoms in Sprague-Dawley (SD) rats or NOD/SCID mice [152
It is worth mentioning that Hansen and colleagues described that Hodgkin lymphoma (HL)-derived CD30+
small EVs induce the release of the pro-inflammatory cytokine IL-8 by healthy eosinophil-like EoL-1 cells and primary granulocytes [153
Although further investigation is required, the typical antitumor activity of neutrophils appears to be easily subverted to a pro-tumorigenic one, by decreasing their cytotoxic ability and potentially increasing their involvement in inflammation.
3.8. Myeloid-Derived Suppressor Cells
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells initially reported to hamper immune responses during chronic infections [154
]. In cancer, these expanded myeloid cells contribute to tumor progression, immune evasion, and provide support to stroma [155
]. Expansion of MDSCs is strongly depending on the ability of the tumor to secrete myeloid-influencing factors, such as IL-6, vascular endothelial growth factor (VEGF), PGE2 and granulocyte-macrophage colony stimulating factor (GM-CSF) [157
Purified MDSCs were shown to inhibit both CD4+
T cell responses in vitro [161
]. Further studies demonstrated that MDSCs down-regulate T cell functions and promote tumor metastasis by secreting a wide array of chemokines [162
]. It has been suggested that the suppressive functions of MDSCs could have been promoted by HSP72 expressed at the surface of lymphoma-derived small EVs which activate STAT3 pathway and stimulate the production of IL-6 in a TLR2/MyD88-dependent manner [137
Wang et al. showed that bone marrow stromal cells (BMSCs) from the MM microenvironment are able to release small EVs which are taken up by MM cells and MDSCs. In this context, BMSC-derived small EVs directly support the survival of MDSCs through a greater activation of STAT1 and STAT3 pathways in vitro. Indeed, activated MM-MDSCs acquire an enhanced T cell suppression activity in vivo which facilitates immune escape of the MM cells [164
MDSCs are one of the most powerful population of cells involved in the siege of the microenvironment. Due to the strong pro-tumorigenic potential of MDSC, tumor cells proliferate undisturbed thanks to the effective immune evasion and nutrient support.
An overview of the hematological malignancy-derived small EV functions based on the content and immune cells is summarized in Table 1
Cancer is a complex disease which doesn’t involve only tumor cells but also a composite cellular microenvironment. Through the multiple strategies and tools deployed by cancer cells to gain proliferative and survival advantages, small extracellular vesicles are one of the most concealed. These vesicles are commonly released by all cells and they are typically used by the cells to communicate with each other. In cancer, small EVs are used to overload the surrounding tissues with pro-tumorigenic signals, derived from TEVs but also from microenvironment cells modified by TEVs.
In this review, we presented how hematological malignancy-derived small EVs possess extremely high potential to re-educate normal tissues, and thus, to re-shape the surrounding tumor microenvironment.
In an initial hostile microenvironment, tumor cells need to alter the normal tissue cell composition to establish a proper niche which will be necessary for cancer growth. Immune cells are the first line of defense against aberrant cells escaped from molecular regulators. Hematological malignancy-derived small EVs actively hijack the immune system guaranteeing a more rapid and successful cancer development. Immune effector cells possess the ability to eradicate cancer cells, thus TEVs are used with the aim to eliminate such threat reducing function, proliferation and migration of effector cells. Hematological malignancies, such as lymphomas and CML, directly target NK cells with small EVs containing molecules which reduce or completely block the cytotoxicity [130
]. A similar strategy is used by DLBCL-EVs to directly regulate immune checkpoint receptor expression or induce apoptosis in T effector cells [55
Cell polarization is another process driven by TEVs to mine the natural immune functions. Through polarization, TEVs change the behavior of certain highly plastic cells, such as monocytes and macrophages, making them gain specific pro-tumorigenic phenotype and function. Under CLL small EVs, monocytes are subjected to polarization that causes changes in immune checkpoint composition, leading them to block T cells activity, and release of pro-inflammatory cytokines [138
]. The latter is also induced by macrophages upon CML-derived small EVs uptake [144
]. Inflammation has an essential effect in the tumorigenesis as it co-participates in reshaping the microenvironment, supporting tumor growth and favoring gene instability. To guarantee a local degree of inflammation is a key feature of cancer and is also an essential process necessary to establish and maintain pre-metastatic niches.
An effective strategy to enhance the bypass of the immune defenses is to hit also regulatory cells which aim to maintain effector cells aware and active. Through the use of small EVs, T lymphoma blocks the maturation of essential patrolling cells such as DCs [121
] making them incapable to stimulate T cell and inducing their differentiation into MDSCs. MM-derived small EVs, in the other hand, directly target MDSCs leading to their expansion and a switch towards pro-tumorigenic phenotype [164
]. Rather than decrease MDSCs activity, MM uses MDSCs immune regulatory ability to inhibit functions of essential effector cell such as CD4+
and NK cells.
Essential for a properly regulated immune response, Bregs and Tregs are used by the tumor to enhance the immune suppression of an already lowered immune system. Lymphoma-derived small EVs were described to cause a persistent activation and expansion of Breg via increased release of IL-10, this causes a deep depression in function and proliferation of effector cells, together with expansion of Tregs [97
Deregulation of both effector and regulatory immune cells through the use of small EVs is an elegant and efficient strategy to hijack immune defenses and allow cancer progression.
Stroma composition evolves in parallel to cancer growth and progression. Indeed, communication with stromal cells allows to generate an appropriate niche surrounding the primary tumor, and potentially in secondary organs, to proliferate and welcome metastatic cells, respectively.
During hematological malignancy development and progression, bones lose the structural protection typically attributed to this tissue. During these processes there is a progressive increase in osteoclasts and decrease in osteoblasts, which break down and produce new bone, respectively. MM is known to continuously modify the cellular component and structure of bone. MM-derived small EVs have been described to transfer molecules responsible for pre-osteoclast rapid differentiation leading to osteoclast expansion and activation [232
]. In turn, osteoclasts release high amount of cytokines and growth factors which ultimately increase cancer cell proliferation and survival [232
]. The increase in osteoclasts causes a progressive bone loss which is further enhanced by the decreased amount of osteoblasts. In line with this, MM-derived small EVs impact on osteoblast function and differentiation, overall leading to osteolysis and a more rapid dissemination of MM cells in the organism [237
]. Bone reshaping by MM-derived small EV is not exclusively given by a direct transfer of molecules into the osteoblasts, but also by reducing MSC differentiation potential [235
Due to their stem cell like properties, MSCs can differentiate in various cell types. This property is used by tumor cells to increase immune response depression, local and distant inflammation and increase tumor pro-survival, proliferation and invasion signals. As previously mentioned, MSCs have a strong impact on immune suppression. Depending on the hematological malignancy, small EVs possess specific molecules which cause distinct effects on MSCs. For instance, lymphoma- and MM-derived small EVs induce MSCs to highly support MDSC population expansion [161
]. In the other hand, MSCs targeted by CLL-derived small EVs are characterized by different gene expression and phenotypical changes. Similarly to fibroblasts, CLL-derived small EVs lead MSCs to differentiate into CAFs, causing release of pro-inflammatory cytokines in the TME, together with pro-survival, growth and migratory signals towards the cancer cells [187
]. Once activated by TEVs, MSCs can, in return, release small EVs directed to immune cells to enrich the pool of pro-tumorigenic immune cells and decrease the anti-tumor activity of effector cells [197
In line with this, monocytes activated by MSC-derived small EVs rapidly differentiate in macrophages, increasing the pool of TAM in the microenvironment. Accumulation of TAMs from MSCs is also correlated with an increase in angiogenesis [139
]. The formation of new blood vessels, allowing nutrients and oxygen uptake, is another essential process required for tumor development and, in advances stages, tumor cells dissemination. Different hematological malignancy-derived small EVs contain various pro-angiogenic factors which trigger ECs proliferation, survival and tube formation [208
Through the release of small EVs, tumor cells can interact with an impressive number of cells, tissues and structures. It is important to stress how the understanding of small EV role during tumor development and progression is essential in order to develop effective anticancer strategies. Achieving a deeper knowledge of this intricate communication system would allow us to identify its weaknesses, as well as to use it as a potential drug delivery strategy.