Peripheral Blood Cell Interactions of Cancer-Derived Exosomes Affect Immune Function

Abstract

Cancer-derived exosomes are constitutively produced and secreted into the blood and biofluids of their host patients providing a liquid biopsy for early detection and diagnosis. Given their ubiquitous nature, cancer exosomes influence biological mechanisms that are beneficial to the tumor cells where they are produced and the microenvironment in which these tumors exist. Accumulating evidence suggests that exosomes transport proteins, lipids, DNA, mRNA, miRNA and long non coding RNA (lncRNA) for the purpose of cell-cell and cell-extracellular communication. These exosomes consistently reflect the status as well as identity of their cell of origin and as such may conceivably be affecting the ability of a functional immune system to recognize and eliminate cancer cells. Recognizing and mapping the pathways in which immune suppression is garnered through these tumor derived exosome (TEX) may lead to treatment strategies in which specific cell membrane proteins or receptors may be targeted, allowing for immune surveillance to once again help with the treatment of cancer. This Review focuses on how cancer exosomes interact with immune cells in the blood.

Introduction

Exosomes are small 30–150 nm sized, extracellular vesicles (EVs) important in the intercellular communication between cells [1–4]. They belong to the nanovesicle family and originate from the intraluminal vesicles of the late endosome, named multivesicular bodies. The fusion of these multivesicular bodies with the cell membrane results in the release of exosomes into the extracellular space. Communication can occur both by transfer of nucleic acids and proteins, or by binding cell-surface receptors and inducing cell signaling pathways [5, 6]. Both normal and tumor cells release exosomes, although TEX have been the subject of a wide range of studies. Exosomes have been shown to be involved in many aspects of the tumor microenvironment (TME) including immune suppression [7, 8], antigen presentation [9–13], a means of acquiring chemotherapeutic resistance [14–18], as biomarker reservoirs [19–25], inducers of angiogenesis [26–28], and vehicles of niche preparation for metastasis [29–33] (Fig. 1). However, modes and mechanisms of uptake are not completely understood. Cells appear to internalize EVs through several endocytic pathways, including clathrin- and caveolin-dependent endocytosis, phagocytosis, and lipid raft–mediated internalization. It is likely that cells utilize multiple routes to take up exosomes, depending on the proteins, glycoproteins, and lipids found on the surface of the vesicles and the target cell itself [34]. Numerous studies show proficient uptake of exosomes by endothelial cells [35–37], epithelial cells [38], fibroblasts [39], myeloid precursors in bone marrow [32, 37], mesenchymal stem cells [37], and other tumor cells [40] (Fig. 2).

Fig. 1. Tumor derived exosomes (TEX) function in favor of metastasis, support angiogenesis, confer chemoresistance and promote immune-suppression and cellular proliferation.

Exosomes from tumor cells were found to release functional biomolecules into the tumor microenvironment thereby affecting the biology of cancer

Fig. 2. Cancer cells release exosomes which are taken up by other cancer cells, endothelial cells, epithelial cells, fibroblasts, bone marrow myeloid precursor cells, and mesenchymal stem cells.

Exosomes from tumor cells were found to release functional biomolecules (protein, RNA, miRNA) into many cell types

Cancer and Exosomes

Cancer cell uptake of exosomes has been well documented and studies even show that exosomes can preferentially associate with cancer cells [41, 42]. Endocytic pathways are utilized by ovarian cancer cells to internalize exosomes from the SKOV3 ovarian cancer cell line [43], by glioblastoma cells [44], and by bladder cancer cells as demonstrated by dose and time dependent uptake of PKH26 labeled bladder exosomes [40]. Treatment with heparin can partially block the active and specific mechanism of uptake, implicating receptor-mediated endocytosis involving heparan sulfate proteoglycans (HSPGs) [40]. HSPGs were also shown to be critical in the internalization of glioblastoma exosomes by glioblastoma cells [45]. Other tumor types have demonstrated exosome uptake, such as colorectal cancer exosomes into lung cancer cells [46] and breast cancer exosomes into breast cancer cell lines [47, 48], although mechanisms underlying the uptake were not addressed.

Diffuse large B cell lymphoma (DLBCL), an aggressive form of lymphoma representing over 40% of adult lymphoma patients, has not until recently been investigated. In an attempt to close the gap in knowledge concerning lymphoma TME immunosuppression, normal human peripheral blood leukocytes were treated with PKH67-labeled lymphoma exosomes and monitored for uptake by measuring fluorescence at different time points using flow cytometry and fluorescent microscopy. Results show that of the four populations examined, B cells and monocytes demonstrated uptake of PKH67 labeled lymphoma exosomes, while T cells and NK cells displayed significantly less uptake [49].

Immune Influence

Macrophages

As exosomes have exhibited multiple forms of influence within the immune system, immune cells have also been investigated regarding their ability to interact with exosomes. Macrophages exhibit specialized capacity for internalizing exosomes, although they typically reside within tissue, not in circulation. Their blood counterparts, monocytes, also appear to have a relatively high level of exosome internalization. Consequently, numerous investigations have focused on exosomal interactions with these myeloid-derived cell types. Macrophages have been shown to internalize exosomes from both normal [50] and malignant sources [51], with differing effects. Macrophages exposed to breast cancer exosomes, but not normal exosomes, activated NF-κB pathways and released pro-inflammatory cytokines like IL-6 and TNFα, possibly due to TLR2 interacting with palmitoylated protein ligands on the exosomes [51]. There are reports of multiple ways macrophages use to interact with exosomes, such as CD169 (SIGLEC) to bind exosomes expressing sialic acids, as seen with B cell-derived exosomes expressing α2, 3-linked sialic acids [52]. Macrophages also utilize a dynamin-dependent phagocytic pathway to internalize vesicles [6, 53]. Leukemia exosomes were found to be efficiently internalized via phagocytosis by macrophages, while non-phagocytic cells such as T cells show few internalized vesicles, with most interaction being with surface-attached vesicles [6].

Monocytes

Monocytes also utilize phagocytic mechanisms to internalize exosomes, perhaps relying on tetraspanin targeting, as was shown by Rana et al. [36] Vesicle internalization by monocytes can induce changes such as production of cytokines like TNFα, which has important downstream ramifications on T cells [54]. Like macrophages and monocytes, other cells of the myeloid lineage such as neutrophils and dendritic cells (DCs) have the ability for exosome uptake. As one of the major infiltrators of the TME, neutrophil interactions with exosomes have been of interest. Investigations in leukemia demonstrate communication between tumor cells and neutrophils, transferring genomic DNAs (gDNAs) of the BCR/ABL hybrid gene from K562 cells to normal neutrophils [55] and even promoting leukemia tumorigenesis in rats [56].

Dendritic Cells

Early investigations in the exosome field recognized follicular DCs as interactors with exosomes [57], and even though no specific receptors had been demonstrated yet, it seemed that alpha 4-integrin on B cell-derived exosomes was important [58]. Integrin complexes with CD9 and CD81 tetraspanins, externalized phosphatidylserine (PS), and CD11a (LFA-1)/ICAM-1 interactions all participate in the binding and uptake processes of DCs [57, 59]. Uptake can be through endocytic mechanisms [60], including phagocytosis [61] and DCs may be even more efficient than macrophages at picking up exosome-sized particles [62, 63]. DCs are affected by their interactions with vesicles. Uptake of mast cell exosomes can induce bone-marrow precursors to acquire antigen presenting capacity to T cells [64], and CD11b + and CD11c + cells in mice began releasing IL-6 and TNFα and upregulated CD86 and MHC class II after exposure to exosomes [63]. Uptake of tumor exosomes by bone marrow precursor cells can inhibit maturation and promote induction of myeloid-derived suppressor cells [65, 66].

B Cells

In addition to APCs like macrophages and DCs, B cells are also capable of internalizing exosomes. B lymphocytes interact with exosomes containing MHC class II and ICAM-1 from mature DCs and obtain the ability to prime naïve T cells and trigger antigen-specific effector responses [67, 68]. B cells may need specific surface proteoglycans (HSPGs) such as syndecans and glypicans to aid exosome uptake. It was demonstrated that chronic lymphocytic leukemia (CLL) exosomes can be internalized via active uptake by various benign cell populations found in the TME such as endothelial cells, myeloid cells, bone marrow mesenchymal stem cells, and even some leukocytes. However, CLL B cells themselves did not show uptake of labeled exosomes – possibly due to a lack of surface HSPGs [37] or syndecan-1 [69]. Additionally, lymphoid cell differentiation from the pre-B-cells in the bone marrow to the plasma cells that produce and release antibody, and the multiple stages of development in between are accompanied by different syndecan-1 protein levels [69]. Syndecan-1 is present on the Pre-B cells and also on the plasma cells but it is absent on the circulating B cells. What this indicates is that syndecan-1 is needed when cells require tissue environments and interactions [70]. These studies support Syndecan-1’s importance to exosome uptake and why CLL B cells, lacking syndecan-1, are unable to, by themselves, uptake exosomes. In a separate study, malignant B cell exosomes showed a natural specificity for B lymphocytes while in another B cells are selectively targeted by exosomes carrying the EBV envelope glycoprotein gp350 [71]. Furthermore, the interaction between an EBV-transformed B cell line (LCL1)-derived exosomes and peripheral blood B cells could be blocked efficiently by anti gp350 antibodies and by anti-CD21 [71]. Mantle cell lymphoma (MCL) exosomes were efficiently internalized by both healthy and diseased B-lymphocytes utilizing a cholesterol dependent pathway independent of clathrin and caveolin [72]. Very little uptake was recorded in bone marrow stroma cell lines, T-cell leukemia cells, or NK cells.

Exosomes have been relatively well-studied in EBV-positive transformed human B cell lines, as these cells constitutively produce large numbers of MVBs and MHC class II molecules [73–75]. The WSU-DLCL2 B cell lymphoma cell line used in our own study [49] as a source of exosomes, is EBV-free. This may be of interest because the virus has been known to hijack and alter exosomes in infected cells. The internalization and subsequent effects of these exosomes may involve viral factors, such as latent membrane protein (LMP). One group examined epithelial uptake of exosomes from EBV-infected B lymphocytes and found uptake was through a dynamin and caveolae-dependent process. In addition, type III latency-derived exosomes were able to induce proliferation and upregulation of ICAM-1 in recipient cells [76]. LMP-1 was also harbored on exosomes from a Burkitt’s lymphoma cell line, and could mimic CD40 signaling to induce stimulatory changes in the B cells that efficiently bound them [77].

T Cells and NK Cells

LMP-1 can also produce an immunosuppressive effect by inhibiting T cell proliferation and NK cytotoxicity [78] and has been shown co-localized with MHC-II on exosomes. Though these studies appear supportive it is still unclear whether T cells can truly obtain antigen/MHC signals from exosomes or EV’s. Dendritic cells have been shown to require dendritic cell derived exosome or DEX’s in order to activate T cells [79, 80]. Specifically, these studies show that in addition to carrying antigen, exosomes promote DC exchange of functional peptide-MHC complexes [80]. T cells have been shown in defined conditions to be activated by antigen presenting cell (APC)-secreted exosomes. Under physiologic conditions however, T cell activation required simply contact with a viable APC. However, further supporting the co-stimulatory scenario is the fact that T cells, in order to be activated, must make contact with B7, ICAM-1 CD28 and LFA-1 [81]. What this might mean to exosome stimulation of T cells is that the signals are weak at best, or that specific requirements such as high MHC density and the presence of ICAM1 and B7 are critical.

There have been few studies investigating uptake of exosomes by peripheral blood cell populations. However, rat pancreatic adenocarcinoma exosomes could be taken up by all leukocyte subpopulations examined, with CD11b + cells demonstrating higher internalization than T or B cells [82]. At this time there is only one other publication addressing peripheral blood uptake of lymphoma exosomes - a study by Hazan-Halevy et al., looking at MCL exosomes and their preferential uptake by B-lymphocytes [72]. In this study, it was shown that exosomes isolated from a MCL cell line, when administered to B lymphocytes, NK cells, and various T lymphocytes, preferentially internalized into B lymphocytes.

While effector cells such as T cells and NK cells are less equipped to internalize vesicles, there is still evidence for a variety of interactions with exosomes. T lymphocytes are affected by exosomes from APCs harboring antigen in MHC class I and II molecules, and constitute an important aspect of immune system communication [73, 83]. The mechanisms of T cell internalization or binding of exosomes from APCs have been posited to involve the T-cell receptor (TCR), CD28, and LFA-1 (CD11a) [84]. Activated T cells use LFA-1 (leukocyte function-associated antigen-1) for binding of DC exosomes containing MHC class II [85]. This LFA-1/ICAM interaction is critical for priming of naïve T cells by exosomes from mature DCs [67]. CD4+ T cells can internalize DC exosomes and stimulate antigen-specific CD8+ CTL while overcoming Treg suppression, with a resultant shift in immune responses [11]. In contrast to T cell priming effects of exosomes, tumor exosomes (TEX) can suppress T cells. Surface ligands such as TRAIL, PD-L1 and FasL result in exosome-mediated cell death [86, 87].

Evidently, despite low exosome internalization, T cells are still subject to exosome-mediated effects. Likewise, even with little uptake, NK cells are influenced through exosomal interactions. NK cell cytotoxicity is frequently seen to be inhibited after exposure to exosomes derived from solid tumors and even EBV-immortalized B cells. It has been surmised that MICB and TGF-β1 expressed on exosomes are responsible [88–90]. One mechanism is through the downregulation of NK activating receptor NKG2D, as exemplified by plasma exosomes from AML patients [91]. In contrast to tumor derived exosomes, dendritic cell derived exosomes can promote NK activation and proliferation through copresentation of NKG2DL with IL-15Ra [92]. Some studies have found evidence of NK cell uptake of exosomes in a time dependent fashion, perhaps utilizing PS located on vesicle membranes as demonstrated in ovarian cancer model [93].

Conclusion

Exosomes are important mediators and regulators of cellular communication. Although they are involved in active immunosuppression, they can also facilitate tumor progression and are also a good source of tumor antigens. However, until a more full understanding of the interplay between the tumor microenvironment and the exosome occurs, effective strategies to mobilize the immune system as an effective anticancer modality will be limited. Recognizing the luminal and surface contents of the exosome is not enough to design exosome-associated therapy but understanding the communication patterns and types of communication (luminal protein delivered or surface to cell protein/protein interaction signaling) will be key. Moreover, identifying which exosome populations are communicating and which are providing additional ligands or receptors in order to facilitate communication will prove necessary to potentiate the immune response.

Funding

Research reported in this publication was supported by NIH awards P20MD006988.