Stem cell-derived exosomes: roles in stromal remodeling, tumor progression, and cancer immunotherapy

The biogenesis of exosomes follows the endocytic-exocytic pathway, which involves various cellular compartments, such as early and late endosomes, lysosomes, and multivesicular bodies (MVBs). The precise mechanisms of biogenesis are not fully understood, but it is well known that sorting of exosomal cargo is a tightly regulated process that occurs under the influence of different sorting machineries (for the detailed mechanisms, see [20]). Once exosomes are sorted, they are released outside the cell following the fusion of MVBs with the plasma membrane.

The formation and secretion of exosomes by biologically active cells are arguably context-dependent, i.e., arising in response to repair processes or tumor progression. MSC-derived exosomes are originated from endocytosed lipid-raft microdomains [54], whereas the endocytic-exocytic pathway may play a role in stem cell differentiation through the release of membrane vesicles containing stem cell markers, such as prominin-1/CD133 [55]. Upon their release from stem cells, secreted exosomes participate in the maintenance of stem cell hierarchies (Fig. 1). Notably, the knowledge about the mechanisms of the biogenesis and origin of exosomes from stem cells could enhance our understanding of exosomes’ functional relevance and could aid in the development of strategies for engineering exosomes for prospective therapeutic applications.

Secreted exosomes exhibit fundamental paracrine mechanisms that mediate cell-to-cell communication through direct receptor-mediated stimulation of target cells and the horizontal transfer of genetic material [22, 38, 56]. It has been proposed that the exosome-mediated exchange of genetic information could be an integral element of the continuum model of stem cell biology, in which the differentiation decision of stem cells is conditioned by cell cycle transit and possibly by environmental stimuli [57]. In this context, as paracrine factors, stem cell-derived exosomes might induce phenotypic changes in recipient cells through genetic exchange and might generate a functional link between stem cells and resident tissue under various physiologic and pathologic conditions.

Intriguingly, exosomal mRNA transcripts for pluripotent transcription factors such as Nanog, Oct-4, HoxB4, Rex-1, and the stem cell-specific Wnt-3 protein, all expressed in stem cell-derived exosomes, may represent several features of stemness, such as maintenance of self-renewal properties and expansion of progenitor cells [21]. Several studies have indicated that stem cell effectors such as WNT [58, 59], Hedgehog [60], and β-catenin [61], which are expressed on stem cell-derived exosomes, may play a potential role in maintaining stemness (Fig. 1). Importantly, several other reports have clearly indicated that stem cell signatures expressed on exosomes play a vital role in maintaining stem cell characteristics, such as differentiation, self-renewal, and maturation [55, 62–65].

In fact, stem cell-derived exosomes may represent several features of their cells of origin and may play a role in gain-of-function properties, genetic instability, and consequent malignant transformation as well as the induction of reparative programs within injured tissues.

Exosomes released from MSCs are a newly discovered source of regeneration in injured tissues, participating in exosome-mediated transfer of regenerative molecules to target cells in a paracrine fashion. For example, stem cell-derived exosomes participate in the suppression of inflammatory responses through the inhibition of hyperproliferative pathways, thus restoring bioenergetics to promote cell survival [66–69]. Moreover, these exosomes may have the potential to reduce infract size and ameliorate heart functions through promoting neovascularization [70–73]. MSC-derived exosomes appear to have multi-pluripotent effects on neurovascular plasticity in rats during traumatic brain injury by promoting endogenous angiogenesis and neurogenesis accompanied by reduced neuro-inflammation [74, 75]. The anti-inflammatory and protective effects of MSC-derived exosomes were also observed during sciatic nerve regeneration in rats that received peripheral nerve cell therapy [76].

Stem cell-derived exosomes may play a role in accelerating the morphologic and functional recovery of the liver through exosome-mediated horizontal transfer of specific mRNA subsets [77], activation of proliferative and regenerative responses [78], reduction in hepatic inflammation, and inhibition of the epithelial-mesenchymal transition in the fibrotic liver [79]. Similarly, acute and chronic lung injuries may be recovered through the secretion of exosomes by MSCs, conferring stem cell-like phenotypes on injured cells to promote self-regenerative programs [80, 81]. Interestingly, the protective effects of MSC-derived exosomes are comparable to those of MSCs, as could be observed in renal recovery [82–85] and wound and bone healing [86–88], indicating that exosomes are functional extensions of MSCs.

Exosomes released from MSCs may potentially stimulate cell survival by inducing anti-apoptotic effects in injured cells and by promoting cell proliferation, as has been well explained for cardiomyocytes [73, 89] and injured renal tubular epithelial cells [90–93]. In fact, exosomes transfer anti-apoptotic miRNAs from stem cells to injured tissues and may contribute to cell survival by transcriptionally repressing the expression of apoptotic genes and activating cell survival signaling. Exosomes released from MSCs and iPS cells could deliver cardioprotective miRNAs to cardiomyocytes in vitro, thereby producing cytoprotective effects and subsequently ameliorating heart function [94, 95]. In addition, exosome-mediated transfer of miRNAs to cardiac progenitor cells may play a role in promoting repair programs in the heart [96, 97].

Exosome-mediated transfer of miRNAs from MSCs promotes neural plasticity and functional recovery after treatment of stroke in the rat brain [98] and may also increase neuronal differentiation of neural progenitor cells [99]. Anti-apoptotic effects of MSC-derived exosomes could also be observed during rat pheochromocytoma protection through apoptotic regulation, i.e., through down-regulation of the expression of Bax, accompanied by reduced cleavage of caspase-3 and up-regulation of Bcl-2 [100]. These findings imply that transplantation of MSC-derived exosomes has enormous potential to protect against cerebral injury and possibly other neuronal diseases.

Several lines of evidence indicate the prospective roles of MSCs and their secreted exosomes in initiating tumor phenotypes. For example, in human renal cell carcinoma, a subset of tumor-initiating cells expressing the MSC marker CD105 release exosomes that promote tumor growth [101, 102], probably through enhanced expression of genes associated with cell migration, matrix remodeling, and angiogenesis [102]. Exosomes from adipose-derived MSCs (AT-MSCs) are enriched in distinct patterns of RNAs that may potentially induce adipogenesis, angiogenesis, apoptosis, and proteolysis in recipient cells [103]. Exosomes from gastric cancer-derived MSCs may deliver miRNAs to human gastric cancer cells and promote their proliferation and migration [104]. Recently, it was shown that glioma-associated stem cells produce substantial amounts of exosomes that exhibit glioma stem cell features and that are capable of sustaining the malignant properties of both glioma cells and glioma stem cells, mainly through the release of exosomes [105].

Moreover, exosomes from BM-derived MSCs (BM-MSCs) transport tumor regulatory miRNAs, anti-apoptotic proteins, and metabolites that promote breast tumor growth [106]. Exosomes from BM-MSCs express higher levels of oncogenic proteins, cytokines, and adhesion molecules, which can be transferred to multiple myeloma cells, subsequently modulating tumor growth in vivo [107].

Lately, it has become apparent that stem cell-derived exosomes may contribute to tumor progression due to these exosomes’ ability to activate signaling pathways by expressing exosome-borne growth factors. For instance, exosomes from BM-MSCs appear to enhance the expression of vascular endothelial growth factor (VEGF) through the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway to promote tumor growth [108]. Exosomes from mast cells express and shuttle mast/stem cell growth factor receptor Kit (KIT), a member of the tyrosine kinase family of growth receptors which promotes tumor growth in recipient lung adenocarcinoma cells by activating the KIT-stem cell factor (SCF) signaling pathway [109]. Given that the Wnt signaling pathway is central to stem cell biology [110, 111], exosomes expressing the stem cell marker prominin-1 may play a potential role in Wnt signaling and pro-metastatic activity in melanoma cells [112]. Taken together, these observations indicate that stem cell-derived exosomes potentially express tumor-promoting molecules, such as miRNAs, oncogenic proteins, growth factors, and adhesion molecules, that engage in pro-metastatic and pro-angiogenic activities by activating signaling pathways (Fig. 2).

Intriguingly, MSC-derived exosomes not only have pro-tumor potencies but also can exert negative effects on tumor growth, depending on the conditions, the tumor type, and the stage of development [113] as well as the expression of tumor suppressor molecules. For example, exosomes from BM-MSCs act as negative regulators of the cell cycle and exert inhibitory effects on tumor growth [114]. Moreover, exosomes from BM-MSCs can transfer miRNAs from the BM and promote dormancy in metastatic breast cancer [115]. Breast cancer growth can also be inhibited by MSC-derived exosomes through miRNA-mediated VEGF suppression [116]. Similarly, exosome-mediated delivery of selective miRNAs from human liver stem cells may inhibit hepatoma growth [117]. Katakowski et al. [118] have shown that intra-tumoral injection of MSC-derived exosomes expressing miR-146 could effectively inhibit glioma xenograft growth. MSC-derived exosomes are capable of incorporating and delivering paclitaxel, which can inhibit tumor growth [119], indicating that stem cell-derived exosomes possess the potential for drug delivery to cancer cells.

Exosome-mediated delivery of tumor suppressor miRNAs and targeting of growth-regulatory pathways, such as the Wnt and Hedgehog pathways, as well as angiogenic pathways, such as the VEGF and kinase pathways, could be novel strategies to monitor tumor growth (Fig. 3). For example, the potent signaling axis miR-140/SOX2/SOX9, which regulates differentiation, stemness, and migration, could be targeted to obstruct tumor progression [120]. Similarly, exosomes from MSCs could be effective in inhibiting bladder tumor cell growth by down-regulating the phosphorylation of Akt kinase [121], whereas exosome-mediated targeting of the VEGF pathway could offer a novel strategy to inhibit tumor growth by inhibiting angiogenesis [116]. However, it remains an open technical challenge to monitor the complex stromal network and to target these pathways within the dynamic tumor microenvironment.

Despite improvement both in the surgical procedures for organ transplantation and in cell-based therapies over the last decade, current methods present potential complications (for example, an increased risk of infection, toxicity, and graft rejection). In this context, exosomes may offer a nontoxic framework of protective molecules during transplantation, as they are capable of inducing endogenous regenerative programs in damaged tissues.

Compared with traditional stem cell therapies, exosome-based cell-free therapy may improve patients’ outcomes considerably, reducing the complications of cell-based therapy. However, steering traditional stem cell-based therapy toward exosome therapy will need further studies. Intriguingly, the combination of exosome-based therapies with existing traditional approaches could improve the therapeutic benefits. The intriguing advantages of stem cell-derived exosomes in therapy are largely due to exosomes’ ability to stimulate endogenous repair processes within the injured tissue and their efficient regulation of immune tolerance. Therefore, the administration of exosomes for prospective therapeutic applications may help to minimize the potential safety risks associated with cellular therapy or transplantation surgery, preventing graft rejection. Manipulating the expression of exosomal contents related to angiogenesis, growth, proliferation, and self-renewal could potentially improve blood flow recovery, may maintain tissue growth, and thus could ultimately ameliorate organ function.

Strikingly, haploidentical MSC transplantation could efficiently postpone the rejection of mismatched skin grafts in patients with severe treatment-resistant acute graft-versus-host disease (GVHD) [154], whereas immunosuppressive exosomes could potentially prevent graft rejection [155]. These findings raise the possibility that immunosuppressive exosomes from MSCs could be a promising therapeutic resource for organ transplantation, preventing graft rejection with great efficacy. An example could be seen when an infusion of MSC-derived exosomes significantly enhances survival and delays the rejection of an allogenic skin graft, as observed in mice, with a corresponding increase in regulatory T cells [150]. Thus, the application of MSC-derived exosomes could be a novel therapeutic platform for future clinical consideration due to the fact that MSC-derived exosomes are well tolerated in patients during the treatment of GVHD [156]. Moreover, MSC-derived exosome treatment could efficiently reduce the pro-inflammatory cytokine response in patients’ peripheral blood mononuclear cells in vitro, and the clinical symptoms of GVHD could be improved significantly shortly after the start of MSC-derived exosome therapy [156]. Importantly, the exosome contents before and after BM-MSC treatment may potentially modulate the immune response [157], suggesting that the exosomal contents of MSC products will be a consideration when used in the clinic in the future.

Despite that stem cell-derived exosomes have been employed in clinical practice for tissue repair and organ transplantation, their application in treating cancers seems challenging. The poor survival of cancer patients, which is mainly associated with the late stages of cancer, a lack of early diagnosis, recurrence, and resistance to existing therapies, poses a potential challenge for improving the long-term survival of patients and reducing the risks of mortality. In this regard, exosomes could serve as a promising resource for early diagnosis, enabling timely monitoring of the disease [20], as well as for developing novel cancer therapies alternative to existing therapies [158]. Cancer immunotherapy principally relies on evoking the host immune system to fight against cancer cells, whereas the immunosuppressive tumor microenvironment is considered a major barrier to the effectiveness of cancer immunotherapies [159]. Given that exosomes play a vital role in central tolerance, efficiently stimulating the immune system and potentially reshaping the immunosuppressive microenvironment [160], this potential could be exploited for the use of stem cell-derived exosomes in cancer immunotherapy, serving as a potent resource of antitumor agents. It is even more likely that exosomes will also serve as a measure of disease progression as well as the response to therapy during the course of immunotherapy.

It is well appreciated that the human T-cell response can be modified using viral and non-viral vectors to promote the selective targeting of cancer cells by introducing exogenous T-cell receptors (TCRs) or chimeric antigen receptors (CARs) [161]. In this regard, using exosomes as vectors for introducing TCRs and/or CARs that specifically target cancer cells may raise the novel possibility of using exosomes for cancer immunotherapy. However, to validate this possibility, further studies will be needed.

Because they are free of potential safety risks, are non-toxic, and do not differentiate into unrequired populations, in contrast to CSCs, exosomes could be a promising platform for cancer therapies. Moreover, precise understanding of the involvement of exosomes in CSC biology and their prospective potential in the prediction and enrichment of cancer-initiating activities may present novel opportunities for developing effective therapies against cancer. Importantly, the use of exosomes, and particularly those derived from MSCs, do not raise potential ethical and legal issues, in contrast to other types of stem cells, and especially human embryonic stem cells (ESCs). Of particular note, the cost of exosome engineering and manipulation would be significantly less, which will be an important clinical consideration.

Despite the enormous potential of exosomes in future clinical settings, strategies will need to be developed for the precise and adoptive manipulation of exosomes for effective usage in local transplantation, systemic infusion, and engineering of their clinical benefits for cancer therapy. Moreover, whether the dose-dependent effects of exosomes in cancer model experiments are promising must be determined because increasing the dose of exosomes may produce unwanted phenotypes and may limit the accessibility of their application to a smaller number of patients. A potential challenge in the field exists due largely to the limitations of standardizing the existing technologies for the isolation, characterization, and engineering of exosomes for their prospective therapeutic use. Therefore, a combination of different high-throughput approaches and precisely optimized methods could overcome the potential limitations related to exosomal detection, characterization, and manipulation [20], which may be implicated in biomarker development and possibly in the clinical use of stem cell-derived exosomes in future cancer therapies [162].