Intimate cross-talk between cancer cells and the tumor microenvironment of B-cell lymphomas: The key role of exosomes

Abstract

B-cell lymphomas are composed of tumor cells and the tumor microenvironment, which is conventionally thought to be composed of a mixture of stromal cells, blood vessels, immune cells, and non-cell components, such as extracellular matrix, cytokines, and chemokines. Exosomes, small endocytically derived vesicles that have been proved to be present in a variety of tumor niches and involved in mediating cell signaling networks, are increasingly regarded as important components of tumor microenvironment. In this review, we first focus on the biogenesis, biodistribution, transportation, and other general characteristics of exosomes and then highlight the vital roles of exosomes in lymphomagenesis and disease progression, particularly from the perspective of immune dysfunction, virus infection, and therapeutic resistance mechanisms.

Keywords

Microenvironment exosomes B-cell lymphomas lymphomagenesis drug resistance immunotherapy

Introduction

Lymphomas are a group of malignant tumors of lymphoid tissues.¹ The World Health Organization (WHO) classification,¹ published in 2016, has categorized lymphomas into more than 80 distinct entities, principally on the basis of morphology, immunophenotype, genetic alterations, and clinical features. Among them, B-cell lymphomas are characterized by arising from malignant transformations of B-lymphoid cells, including Hodgkin’s lymphomas and most of non-Hodgkin lymphomas. The highly complex process of lymphomagenesis and tumor progression may involve chromosomal abnormalities and gene mutations, as well as the dynamic interaction between the tumor cells and the tumor microenvironment (TME), which has profound influence on cell behaviors physiologically and pathologically.^2,3

The TME of B-cell lymphomas mainly contains variable numbers of mesenchymal stem cells, stromal cells, immune cells, and, but not limited to, soluble factors.^4,5 The complex interplay between tumor cells and TME regulates tumorigenesis and provides novel targets for immunotherapies. One of the most promising approaches to activating therapeutic antitumor immunity is the blockade of immune checkpoints, such as the antibodies of cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programed cell death-1 (PD-1).⁶ According to 56th annual meeting of the American Society of Hematology, Phase-I clinical trials have confirmed the activity and safety of nivolumab⁷ and pembrolizumab⁸ (both drugs are monoclonal antibodies that bind to and inhibit PD-1) in patients with lymphomas. The mechanism of immune checkpoint inhibitors (ICIs) for tumor therapy is to re-activate antitumor immune system by blocking inhibitory immune checkpoint molecules or their ligands, which are often overexpressed on immune cells of TME and tumor-cell surfaces.^9,10 PD-1 is often overexpressed on surfaces of T cells in TME, leading to suppression of T-cell proliferation and function and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production.⁹ Nivolumab, an IgG4-κ immunoglobulin of about 146 kDa that binds to PD-1, promotes CTL proliferation and resistance to Treg-mediated suppression and consequently reverse T-cell exhaustion.^11,12 Despite clinical successes to date, the exact mechanisms of ICIs are not fully understood.

Recently, it has been increasingly noticed that the potential functions of extracellular vesicles, such as microvesicles (MVs) and exosomes, contributed to various aspects of tumorigenesis,^13,14 particularly with a focus on the exosome-mediated cell signaling. The cancer cell–released exosomes mediate cell–cell interactions and the communications between neoplastic cells and non-cell elements, which may also play a fundamental role in building an organization of the TME.¹⁵ Exosomes affect tumor development and progression mainly by altering the phenotype of receiver cells via diverse exosomal cargoes including proteins, DNA, messenger RNAs (mRNAs), and microRNAs.¹⁶

This review will summarize recent advances on the fundamental understanding of exosomes and the TME of B-cell lymphomas and uncover the interaction between them based on published experimental evidences.

Exosomes’ biogenesis, biodistribution, and transportation

Exosomes, a group of typical microparticles (30–100 nm in size) secreted by different cells, are first reported by Pan and Johnstone¹⁶ in 1983 as unwanted cellular components extruding from reticulocytes. Nowadays, exosomes are recognized as the natural carriers of many signal molecules that mediate cell–cell communication and exert critical influences on tumorigenesis,¹⁷ autoimmune diseases,¹⁸ infections,^19,20 and cardiovascular diseases.^21,22

The biogenesis of exosomes starts with the formation of late endosomes, also called multivesicular bodies (MVBs), which originate from reverse invagination of the cellular plasma membrane. Exosomes are released from cells into adjacent microenvironment when the peripheral membrane of MVBs fuses with the cellular plasma membrane.²³ Mazzeo et al.²⁴ showed that protein kinase D1/2 participated in this complex process, including the maturation of MVBs and the secretion of exosomes in T and B lymphocytes. A wide variety of cells are capable of releasing exosomes, including mesenchymal stem cells (MSCs),²⁵ dendritic cells (DCs),²⁶ B cells,^27,28 T cells,^29,30 natural killer (NK) cells,³¹ and particularly tumor cells.³² Thus, apart from blood, lymph, and cerebrospinal fluid, exosomes are also present in various other body fluids including urine, synovial fluid, breast milk, saliva, amniotic fluid, and malignant effusions of ascites. Once released, exosomes can spread through body fluids to affect functions of the recipient cells.³³ B-cell lymphomas can involve almost any lymphoid tissue in the body, including lymph nodes and different organs and occasionally involving bone and causing osteolytic lesions.³⁴ It is more likely to spread to distant parts of the body partly because the lymphatic system is composed of a network of interconnecting lymph nodes and lymphatic vessels that assist tumor cells to spread distantly. Peinado et al.³⁵ reported that exosome production, biodistribution, transportation, and subsequent function of educating bone marrow progenitor cells assist melanoma growth and metastasis. We assume that exosomes derived from lymphoma cells also participate in the lymphoma dissemination, but the detailed mechanism needs to be further studied. The exact mechanism of how exosomes communicate with target cells is not yet clear. Based on in vitro studies, it is proposed that exosomes might mediate distant intercellular communication through membrane fusion,³⁶ endocytosis,³⁷ or receptor-mediated internalization.^38–40 In addition, accumulating evidence points out a new mechanism that exosomes burst and release their contents to regulate the recipient cells, similar to a paracrine mechanism.⁴¹

Composition and cargo

Exosomes are generally composed of two parts, the bilayer lipid membrane and the intra-vesicular content that is closely related to the reverse invagination of the plasma membrane.⁴² These nanoscale membrane vesicles have powerful effects in regulating physiological as well as pathological processes in extracellular milieu and rely on intra-vesicular cargoes, including mRNAs, microRNAs, proteins, lipids, and signaling molecules. Recent research showed that the presence of single- and double-stranded DNA molecules also can be detected in tumor-derived exosomes.^43,44 Dong et al.⁴⁵ reported larger quantity of long noncoding RNAs (lncRNAs) in the exosomes from colorectal cancer serum than that in other membrane vesicles, including apoptotic bodies and MVs. Most contents enriched in exosomes are molecules with the same orientation as that of the producer cells, while a small set of cellular components is selectively enriched, including endosome-associated proteins (e.g. Alix), proteins that associated with lipid raft (flotillin and glycosylphosphatidylinositol-anchored protein), and tetraspanins (e.g. CD63, CD9, CD81, and CD82).^17,46 Although these markers play crucial roles in purification and identification of exosomes, these classically used exosome markers are not unique to exosomes, whereas they are similarly present in all EVs.³⁷ As a result of their endosomal origin, exosomes generally do not contain mitochondria, endoplasmic reticulum, or nuclear proteins.⁴⁷ The illustration of biogenesis and composition of exosome and the possible communication mechanism between exosomes and target cells are shown in Figure 1.

Figure 1.

Biogenesis and composition of exosome, and the possible communication mechanism between exosomes and target cells. Biogenesis of exosomes begins with the formation of late endosomes, also called multivesicular bodies (MVBs), which originate from reverse invagination of the cellular plasma membrane. Exosomes are released from cells into adjacent microenvironment when the peripheral membrane of MVBs fuses with the cellular plasma membrane. Exosomes are composed of two parts, the bilayer lipid membrane and the intra-vesicular content, including mRNAs, microRNAs, DNA, proteins, lipids, and signaling molecules. Some proteins enriched in exosomes are selectively enriched, termed as exosomal markers, including Alix, flotillin, CD63, CD9, CD81, and CD82. The secreted exosomes act in autocrine or paracine to exhibit their effects with cells. It is proposed that exosomes might mediate distant intercellular communication though membrane fusion, endocytosis, or receptor-mediated internalization.

Biological functions in tumorigenesis

Exosomes seem to be a novel and powerful intercellular signal mediator between cancer cells and its microenvironment. The functions of these nanosized vesicles appear to rely on its cargoes and surface biomolecules. Multiple studies indicate that exosomes are actually involved in different phases of carcinogenesis and tumor development.^48–55

From the perspective of tumor-initiating property, Melo et al.¹⁷ demonstrated that the non-tumorigenic MCF10A cells formed tumor mass in nude mice when co-injected with cancer cell–derived exosomes, and Dicer blocking in these exosomes could reduce tumor formation. This oncogenic conversion of MCF10A cells attributes to precursor microRNAs (pre-miRNAs), along with Dicer, AGO2 (Argonaute2), and TRBP (HIV-1 TAR RNA binding protein) in exosomes of cancer cells. The functional microRNAs within exosomes are associated with the RNA-induced silencing complex (RISC)-loading complex (RLC) that display capacity of processing pre-miRNAs to mature microRNAs in a cell-independent manner. Moreover, Boelens et al.⁴⁸ showed that RNAs within stromal cell-derived exosomes stimulated STAT1 and NOTCH3 signaling in breast cancer cells and disseminated tumor cells through promoting stem cell population, which is blamed for the initiation of tumor formation and therapy resistance.

In addition to acting as an initiator of carcinogenesis, cancer cell–derived exosomes also can promote tumor-cell dissemination and metastasis via distinct cargo molecules. Pre-metastatic niches usually have been formed in the receptor organs before tumor-cell metastasis and in which exosomes might be involved. Costa-Silva et al.⁴⁹ reported that pancreatic ductal adenocarcinomas–secreted exosomes induced pre-metastatic niche formation due to the exosomal cargo macrophage migration inhibitory factor (MIF). Besides, their subsequent study found that a variety of tumor-derived exosomes uptaken by organ-specific recipient cells also established a favorable microenvironment that promoted the colonization and growth of disseminated tumor cells upon their arrival.⁵⁰ Proteomics uncovered that distinct integrin expression patterns contributed to tumor metastasis, for example, the exosomal integrins α6β4 and α6β1 for lung metastasis and exosomal integrin αvβ5 for liver metastasis. They also reported that exosomal integrin uptaken by recipient cells induced expression of several metastasis-related genes, including pro-inflammatory S100. In the respect of tumor metastasis, Zhou et al.⁵¹ showed that breast cancer–derived exosomes destroyed vascular endothelial barrier to facilitate metastasis by targeting the tight junction protein ZO-1 via exosomal miR-105. Zhang et al.⁵² revealed the role of exosomal cargo in tumor metastasis by conducting the following PTEN experiment. PTEN loss could promote the tumor-cell dissemination in the brain since PTEN is an important tumor suppressor. Intriguingly, the PTEN level in PTEN-loss brain metastatic tumor cells was restored once leaving the brain microenvironment. This brain microenvironment-dependent, reversible PTEN mRNA and protein downregulation were epigenetically mediated by the microRNAs in brain astrocyte-derived exosomes. Thus, depletion of PTEN-targeting microRNAs in astrocytes or inhibition of astrocyte-derived exosome secretion could rescue the PTEN loss and simultaneously suppress brain metastasis. A report showed the result of a reproducibility project: melanoma exosomes facilitated bone marrow progenitor cells transforming into a pro-metastatic phenotype via MET protein, an oncogenic receptor tyrosine kinase that is frequently aberrantly activated in tumors.⁵³ Besides above studies, Syn et al.⁵⁴ have elaborated exosome-mediated metastasis in their study.

In addition to the above-mentioned pathological and physiology functions, exosomes can be applied as effective tumor markers. Melo et al.⁵⁵ showed that the surface of pancreatic cancer exosomes was rich in glypican-1 (GPC1), and GPC1⁺ exosomes could be detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, therefore distinguishing between patients with a benign pancreatic disease and early- or late-stage pancreatic cancer. Their study revealed that the levels of GPC1⁺ circulating exosomes were related to tumor burden and the survival of pre- and post-surgical patients.

Collectively, these studies indicate that the evil little things, cancer-derived exosomes, could promote tumor metastasis and the formation of pre-metastatic niches and may also serve as a potential non-invasive diagnostic marker for the development of tumor. Since few exosome-related studies have been performed on lymphoma, we will discuss recent reports that suggested exosomes play a peculiar role in B-cell lymphomagenesis in the following sections.

Role of the components in the lymphoma microenvironment

B-cell lymphomas consist of a wide range of entities and consequently have various clinical behaviors, causing significant morbidity and mortality worldwide. While substantial proportion of studies indicated that genetic alterations can give rise to lymphomagenesis, like many other malignancies, increasing evidences also revealed that the TME has crucial impact on lymphoma initiation and progression.⁵ The special structures of bone marrow and lymphoid organs, including lymph nodes and the spleen, make the TME of lymphomas significantly different from that of other hematological malignancies or solid tumors. In addition to malignant cells, the TME of B-cell lymphomas is composed of a mixture of stromal cells (fibroblastic reticular cells and mesenchymal stem/stromal cells), blood vessels, immune cells (T- and B-cells, macrophages, dendritic cells, and antigen-presenting cells), and non-cell components, such as extracellular matrix, cytokines, and chemokines. Notably, infectious agents are involved in microenvironmental abnormalities that might result in lymphomas. In return, lymphomas bring changes to the microenvironment.

Mesenchymal stem/stromal cells (MSCs) represent one of the central cellular components of the lymphoma microenvironment⁵⁶ and play an important role in the regulation of tumor engraftment, proliferation, and vessel density⁵⁷ as well as drug resistance.⁵⁸ It is also believed that macrophages are present virtually in any tumor niche, while within the B-cell lymphomas they are named tumor-associated macrophages (TAMs) or lymphoma-associated macrophages (LAM). Both MSCs and LAM contribute significantly to the progression of lymphoma. MSCs induced lymphoma progression mainly by enhancing the growth of transplanted tumors through elevating the expression of CC Chemokine Receptor 2 (CCR2) ligands (e.g. CCL-2, CCL-7, and CCL-12) to facilitate recruiting macrophages/monocytes toward the tumor sites.⁵⁹ Recently, a novel mechanism showed by Lin et al.⁶⁰ have uncovered that MSCs recruited macrophages to the tumor sites via CCR2 chemotaxis axis and subsequently facilitated tumor growth in melanoma and lymphoma under the help of tumor cell–derived exosomes. Therefore, MSCs and LAM, even the tiny particles such as exosomes, might be the potential therapeutic targets for reducing tumor burden.

The intratumoral T-cells, a heterogeneous group of immune cells that are subdivided into various subpopulations including CD8⁺ T cells, regulatory T cells (Treg), helper T cells (Th), and follicular helper T cells (TFH), account for up to 50% of the intratumoral cells demonstrated by tumor biopsies from those patients diagnosed with B-cell lymphomas.⁶¹ The role of T-cell subpopulations in lymphoma progression is obviously complex, which produce great effects not only on immunosuppression^62–64 but also on activation of antitumor immune responses.⁶⁵

Besides, data obtained from immunotherapies also give affirmation to NK cells in the functions of anti-lymphoma.⁶⁶ Natural killer group 2D (NKG2D), a germline-encoded receptor, plays an important role in NK-mediated immunosurveillance, while it is not only expressed on NK cells but also on activated CD8⁺ T cells, natural killer T (NKT) cells, and certain CD4⁺ T cells. NKG2D is an activating surface molecule that recognizes stress-inducible self-molecules, including major histocompatibility complex (MHC)-related ligands. NKG2D is poorly expressed in healthy cells but significantly upregulated in malignant cells.⁶⁷ The overexpression of NKG2D in tumor cells induced the upregulation of NKG2D ligands (NKG2D-L) leading to a higher sensitivity of NKG2D-dependent NK-mediated cell killing.⁶⁸ The study on Eµ-Myc mouse models found an NKG2D-L upregulation at early stages of lymphomagenesis, uncovered a mechanism for oncogenic transformation.⁶⁹ It is believed that the downregulation of NKG2D-L on circulating immune cells suppresses the NKG2D-dependent pathway of cytotoxicity,^70,71 thus it could be identified as a mechanism of tumor escape. Moreover, Salih et al.⁷⁰ indicated that NKG2D-L-expressing tumor cells evaded the immunosurveillance through matrix metalloprotease cleavage of the ligands from cell surface into a soluble form. Recently, plenty of studies showed a novel mechanism for NKG2D/NKG2DL-dependent pathway in immune escape with a focus on tumor-cell exosomes.^72–75

Lymphoma exosomes participate in antitumor effect and immune dysfunction

Understanding how exosomes interact with the immune system in B-cell lymphomas is significantly important for the development of immunotherapeutic strategies. Tumor cell–derived exosomes, which have been reported to contain tumor antigens and MHC class I molecules, can present tumor antigens to dendritic cells and induce CD8⁺ T-cell-dependent antitumor immune responses, leading to direct suppression of malignant tumor development.^76,77 Back in the 1990s, Raposo et al.⁷⁸ have shown that the exosome-mediated fusion between MHC class II and the plasma membrane was the new pathway for antigen presentation. In the study of lymphomas, Chen et al.⁷⁹ showed that exosomes released from heat-shocked mouse B lymphoma cells (HS-Exo) contain heat shock proteins (HSP) and numerous amounts of molecules involved in immunogenicity, including MHC class I, MHC class II, CD40, CD86, RANTES, and IL-1β. Furthermore, HS-Exo could activate T-cell responses more potently and induce significantly increased antitumor immune responses in prophylaxis and therapeutic in vivo lymphoma models. In conclusion, exosomes from particular cells provide innovative approaches for tumor immunotherapy due to their immunogenicity.

However, tumor cell–derived exosomes are also crucial in conferring intercellular signals to regulate the evasion from immunosurveillance and immunosuppression in the TME during the disease progression.^79,80 Exosomes participate in immunoregulation through proteins on cell surface and the intra-vesicular content. Tumor-derived vesicles were proved to induce immune cell apoptosis through the receptor-mediated pathway.^81,82 For instance, previous evidence pointed toward Fas ligand (FasL)-positive microvesicles as the mediator of inhibiting immune responses by inducing T-cell apoptosis.^83,84 Recently, Ahmed et al.⁸⁵ showed that Epstein–Barr virus (EBV), implicated in several types of lymphomas, could hijack the exosomes to induce apoptosis in a large number of cell types, including T-cells, B-cells, and epithelial cells, possibly led to T-cell depression.

In addition to inducing immune cell apoptosis, another way of exosomes assisting cancer cell to escape the immunosurveillance is reducing the antigen presentation. NK-cell activities are regulated by the interplay of activating and inhibitory receptors.⁸⁶ NKG2D has been shown as an important component of the immune network in B-cell lymphomas, and NKG2D/NKG2DL-mediated system in cancer cells are well-documented.^73,75,87 Hedlund et al.⁸⁸ used lymphoma cell line Raji as a model for the studies of NKG2D receptor-ligand system in B-cell lymphomas. They found that NKG2DL-bearing lymphoma exosomes suppressed the NKG2D receptor–mediated cytotoxicity, thus impaired NK-cell function. This study suggested that tumor evasion of immune surveillance was regulated by the shedding of exosomes, which affect the cytotoxic ability of NK cells by the active antigen molecules. The new mechanism might somewhat provide an explanation of the high rate of immune dysfunction in patients with relapsed lymphomas. In Figure 2, we have shown that lymphoma-derived exosomes transfer cargoes to the surrounding cells leading to immune dysfunction.

Figure 2.

Elucidation of the B-cell lymphoma-derived exosome-mediated transformation of the tumor microenvironment. Lymphoma-derived exosomes transfer varied cargoes to the surrounding stromal, endothelial, immune, and lymphoma cells leading to immune dysfunction, stromal cell phenotype transformation, angiogenesis, and therapeutic resistance.

Exosomes participate in multiple kinds of immune-escape mechanisms by which cancer cell evasion takes control over the immune system. Among a variety of complex immune mechanisms, apoptosis induction and impaired antigen presentation have been reported in B-cell lymphomas. Other immune regulations of exosomes, like gene regulatory effects⁸⁹ and secretion of immunosuppressive factors,⁹⁰ are reported in lung cancer and breast cancer, respectively.

Potential roles of exosomes in virus-related lymphomas

Multiple evidences have established the theory that viral agents, including EBV, the Kaposi’s sarcoma-associated herpesvirus (KSHV), and human immunodeficiency virus type 1 (HIV-1), are considered fully responsible for the development of lymphoid malignancies, especially in the initiation of B-cell lymphomas.

Three pathomechanisms implicated in this malignant transformation process have been identified:⁹¹ first, direct tumorigenesis action, such as lymphocyte infection and transformation directly induced by EBV and KSHV; second, inflammatory stimulation, some virus, like hepatitis C virus (HCV), stimulates chronic B-cell activation and inflammation by producing viral antigenic and soluble factors; and third, immunodeficiency, as particularly caused by HIV-1, which facilitates depletion of Th and Tregs, thereby leading to immunosuppression and the initiation of malignant neoplasm.

While the majority of published researches on viral-associated lymphomas were focused on the direct transforming abilities of viral products in tumor cells, there is increasing evidence that the indirect actions (i.e. immunosuppression, neovasculature, and TME components) of different viruses also play significant roles in lymphomagenesis.³ As oncogenic properties, multiple viral agents participate in microenvironmental abnormalities through a wide set of viral genes and their encoded oncoproteins, including HIV-1 p24, EBV EBNAs, LMPs, BARF, KSHV vFLIP, and LANAs.^92,93 Besides the well-known HIV-1 capacity of causing the depletion of CD4⁺ T cells, there are other virus-driven immunodeficiency mechanisms impacting the immune TME. For example, EBV and HIV-1 are proved capable of inducing the overexpression of PD-L1 on antigen-presenting cells, thus resulting in immunosuppression by the increased apoptosis of T cells. Furthermore, macrophages are also involved in viral-associated immunity dysfunction, since the tissue macrophages represent a reservoir of HIV-1, and HIV-positive macrophages have been shown with lymphomagenic potential in severe combined immunodeficiency (SCID) mice.⁹⁴ In addition, Liapis et al.⁹⁵ found that the microvessel density in AIDS-related lymphomas was probably associated with EBV latent membrane protein 1 (LMP1) signaling and revealed a mechanism of virus-induced angiogenesis, showing that virus-infected endothelial cells modified the TME.

Recent studies indicated that KSHV, EBV, and possibly other viruses can manipulate the TME through the secretion of specific exosomes, small endocytically derived vesicles that contains peculiar viral and cellular components. Viral exosomes contain high levels of viral genetic materials, like microRNAs, and oncogenic proteins that both activate crucial signaling pathways in recipient cells.⁹⁶ The human gamma herpesviruses KSHV is associated with primary effusion lymphoma (PEL) and several other lymphoid malignancies.^97,98 Chugh et al.⁹⁹ reported that exosomes derived from patients with KSHV-associated malignancies and KSHV mouse models contained KSHV-encoded microRNAs such as miR-17-92 cluster, which is relevant in numerous signaling pathways that are identified to be critical in KSHV pathogenesis. Moreover, they discovered that exosomal virus-encoded microRNAs contributed to increased cell migration and IL-6 secretion in endothelial cells. Recently, another similar study with a focus on EBV, a virus associated with heterogeneous lymphomas, found that EBV-positive Burkitt’s lymphoma cells Raji released exosomes with miR-155 inducing angiogenesis in remote recipient cells, whereas no major difference was found in co-culture with EBV-negative Burkitt’s lymphoma cells.¹⁰⁰ Thus, it would be reasonable to believe that specific viral exosomal microRNAs contribute to angiogenesis of vascular endothelial cells, subsequently leading to pathophysiologic angiogenesis. Further research is obviously necessary in order to identify whether such nanoparticles within the TME can lead to angiogenesis even carcinogenesis in vivo.

The B-cell stimulatory capacities of exosomes released by EBV-infected lymphoma cells include the production of the activation-induced cytidine deaminase (AID), the expression of circle and germline transcripts for IgG1, and the promotion of a differentiation shift toward plasmablast-like phenotype. The viral LMP1 is proved to contribute to lymphomagenesis, although other viral proteins and cellular factors within the exosomes may also participate in these processes.¹⁰¹ Early studies suggested that LMP1 was capable of increasing the expression of a variety of proteins, including cytoskeletal proteins,¹⁰² adhesion molecules,¹⁰³ and fascin¹⁰⁴ which were relevant with the invasive migration of EBV-transformed cells.¹⁰³ In addition to LMP1 protein, EBV exosomes also contain mRNAs encoding LMP1, LMP2, EBNA1 (Epstein–Barr nuclear antigen 1), and EBNA2 (Epstein–Barr nuclear antigen 2),¹⁰⁵ but the functions of these exosomal mRNAs remain unknown. It is assumed that these exosomal mRNAs probably can be translated into oncogenic proteins in the host recipient cells. Notably, EBV-infected cells released exosomes that contain EBV specifically encoded microRNAs, and these exosomal microRNAs could induce monocyte/macrophage transforming into the tumor-associated macrophage (TAM), which showed correlation with disease outcome. Kotani¹⁰⁶ found that EBV⁺ lymphoma cells were embedded in non-neoplastic bystanders, such as B and T cells and macrophages, and interestingly, the lymphoma cells were unable to be engrafted in immunodeficient mice without these bystander cells. Their study conclusively showed the reason turned out to be that exosomal microRNAs from EBV⁺ lymphoma cells are capable of redirecting bystander cells into the tumor-supportive niche. The researchers treated monocyte/macrophage with exosomes secreted from EBV⁺ lymphoma cells, and thereafter, monocyte/macrophage was transferred into tumor-associated macrophages (TAM), due to CD69, IL-10, and tumor necrosis factor (TNF) which were induced by exosomal EBV-microRNAs.

Recent study involving immune dysfunction indicated that EBV-infected exosomes could induce apoptosis in several different kinds of cells, including T-cells through the Fas-ligand-mediated pathway, leading to the suppression of T-cell signaling.⁸⁵ Baglio et al.¹⁰⁷ showed that virus-modified exosomes also have a physiological role in the establishment of a life-long asymptomatic latent infection. Viral exosome–mediated immunity dysfunction has also been reported in other disorders recently.¹⁰⁸

Collectively, these studies indicated that the lymphoma microenvironment is altered in viral infection conditions, at least leading to angiogenesis, phenotype transformation, and immune dysfunction partly via the secretion of microRNAs and proteins carried by exosomes (Figure 2).

Exosomes as communicators of drug resistance in lymphoma

Drug resistance remains a major obstacle in implementing therapies to achieve successful outcomes against tumor. Recent study showed that exosomes removal might be an adjuvant therapy for malignancies due to the vital roles of these cell-derived microparticles in therapeutic resistance.¹⁰⁹ The intuitive mechanisms of drug resistance involving exosomes might be the sequestration of cytotoxic drugs in the intracellular microvesicles and the subsequent expulsion, to wipe out drug effect within the cells.¹¹⁰ The results of Luciani et al.¹¹¹ are in accord with the above conclusion and demonstrated that lymphoma, adenocarcinoma, and melanoma cells confiscated drugs such as vinblastin, 5-flurouracil, and cisplatin in their endosomal compartments. Further studies revealed that high levels of adenosine triphosphate (ATP)-binding cassette (ABC) transporter A3 (ABCA3) were related to drug resistance,^112,113 especially by drug expulsion which might be modulated by microparticles.¹¹⁴

As for targeting therapy of B-cell lymphoma exosomes, Aung et al.¹¹⁵ supported this mechanism by showing that exosomes, carrying CD20 and lysosome-related organelle-associated ABCA3, released from B-cell lymphoma, bound to therapeutic anti-CD20 antibody and consumed complement, thereby impairing antibody-dependent cell-mediated cytotoxicity (ADCC) and protecting cancer cells from antibody attack. Besides, they also mentioned that more than one-third plasma rituximab bound to exosomes in patients treated with therapeutic antibody for B-cell lymphoma. Additionally, removing exosomes from plasma samples resulted in considerable improvements in the effect of rituximab against lymphoma cell lines and against autologous cancer cells in vitro. In addition to lymphoma, Ciravolo et al.¹¹⁶ also showed that the human epidermal growth factor receptor 2 (HER2)-positive exosomes released by breast cancer cells markedly attenuated the cross-talk between cancer cells and the humanized antibody trastuzumab, leading to tumor aggressiveness and therapeutic interference.

Recently, another study by Oksvold et al.¹¹⁷ has shown a similar observation that B-cell lymphoma-derived exosomes, isolated by magnetic beads, were capable to reduce rituximab-induced complement-dependent cytotoxicity. Taken together, these studies revealed a novel mechanism of drug resistance in lymphoma, which is linked to an ABCA3-dependent pathway of exosome secretion (Figure 2).

The complex interplay between lymphoma cell–derived exosomes and the TME provides target for therapeutic strategies. Targeting cancer exosomes for metastatic cancer treatment proved to be one of the therapeutic methods with great promise.¹¹⁸ Researchers recently reported that depletion of ABCA3 either via using short hairpin RNA (shRNA) or by applying the cyclooxygenase inhibitor indomethacin significantly diminished exosome secretion and increased drug susceptibility.¹¹⁹ These studies potentially facilitate new strategies to overcome drug resistance.

Summary and prospect

The malignant progression of lymphoma is complicated, and initially, the major mechanisms of B-cell lymphomagenesis were focused on the aberrations in primary tumor cells, while nowadays there is a growing recognition concentrated on the role of the TME, a complex community of stromal cells, blood vessels, immune cells, and some non-cell components. In contrast to conventional cytotoxic chemotherapy, emerging immunotherapy is a treatment that mediates patients’ own immune system to trigger antitumor response and has less toxicity. However, TME tends to be the main limitative factor preventing the sensitization and infiltration of effector T cells, thus suppressing antitumor immune response. Therefore, deeper understanding of the interaction between lymphoma cells and TME may help define new treatments or preventive tools. This review has presented a conceptual framework of the lymphoma microenvironment, contributing to cell biology, cell signaling, immunology, and treatment resistance, but not limited to aforementioned aspects. The once-underappreciated components of the microenvironment have been reviewed from numerous publications. Moreover, increasing evidences have placed a spotlight on the role of exosomes in the TME of B-cell lymphomas, especially from perspectives of mediating immune dysfunction, virus infection, and drug resistance. A profound understanding of multifunctional exosomes in the microenvironment will help to illustrate the mechanisms of lymphomagenesis and pave the way for lymphoma diagnosis and targeted therapy.

Although tissue biopsy has been identified as standard diagnostic procedure for tumor diagnosis, there are limitations in tissue biopsy–based procedures, especially for early-stage detection or when biopsy is not easily accessible. In recent years, liquid biopsy exerts a huge fascination on a great many scholars and becomes a prominent research topic in precision medicine for varied cancers. In addition to circulating tumor cells (CTC) as well as circulating DNA (ctDNA) and circulating RNA (ctRNA), circulating exosomes from different body fluid samples have been recognized as potential biomarkers because they deliver enriched cargoes, such as mRNAs, microRNAs, lncRNA, and proteins, which are well protected from nucleases and proteases by the bilayer lipid membrane of exosomes. Although a number of pioneer studies reported some biomarkers in other different diseases,^120,121 exosomal biomarkers of lymphomas and their clinical values are waiting for investigation in the near future. Beyond this, the isolation methods of exosomes, which might impact the overall study, are far from perfect and cannot be used widespread. Thus, it is necessary to develop a rapid and efficient purification method to gain high quality, high purity, and large-scale exosomes instead of mixed extracellular vesicle populations. We should cherish the hope that we will be amply rewarded in tumor therapy if the aforementioned tasks have been solved in the coming future.

Footnotes

Acknowledgements

B.X. and T.W. have contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors wish to thank National Natural Science Foundation of China (Grant No. 81570177) that supported our study.

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