When it comes to therapeutic options, research results are even scarcer. At this point we want to express a warning. Since we are still very early in this research field and the biology behind exosomes is incompletely understood, the application of these new and innovative techniques, although of high research interest, may cause unforeseen harm to patients, and so experiments should be well planned.
5.1. Modulation Of Immune System
An imbalance of the immune system might lead to overaction of the inflammatory pathway. Inflammation gets induced through tissue damage or recognition of pathogens. Chronic inflammation has been associated with an increased risk of malignant cell transformation [60
]. If progression of cancer takes place, different modulations within the immune system are required: apoptosis, proliferation and angiogenesis. The culmination of inflammatory mediators (Stat3, IL-6, TNF-alpha) guides to an immuno-suppression and following progression of cancer. Exosomes are able to participate in this regulation and alteration of immune players with either inhibiting or promoting action.
Exosomes isolated from malignant ascites of ovarian cancer modulated the function of monocytic cells [62
]. Production and secretion of proinflammatory cytokines was induced, like interleukin, tumour necrosis factor alpha via TLR2 and 4, leading to an activated nuclear factor as well as signal transducer and activator of transcription (Stat3). The appearance of activated nuclear factor is often observed in cancer cells and results in a more aggressive phenotype with tissue invasion, metastasis and resistance to growth inhibition. [63
] Communication between Stat3 and nuclear factor is important for regulation among inflammatory and cancer cells. Apoptosis, angiogenesis and tumour invasion is regulated making resistance towards immune surveillance possible [64
Apoptosis, as one of the first steps for cancer progression, can be seen through activated T-cells via expression of death ligands (FasL [65
] and TRAIL [66
]), leading to an evasion of immune surveillance. Concluding modulation with release of FasL-positive exosomes is able to guide immune escape. A regulation of T-cell apoptosis was exemplary shown in human colorectal cancer [67
]. Adding up impaired dendritic cell differentiation and suppression of natural killer cells has a similar effect [68
]. In metastatic ovarian cancer, Yokoi et al. proved that cancer-derived exosomes induced apoptosis after uptake by mesothelial cells perforated the peritoneum resulting in peritoneal dissemination [70
Concerning proliferation as an important modulation, exosomes derived from thrombin-activated platelets showed an exciting potential for survival, proliferation and chemotaxis of hematopoietic cells [71
]. Besides they were capable of activating monocytes and B-cells [72
Angiogenesis plays an important role during tumour growth, supplying oxygen and nutrition to the cancer and showing effect on an exosomal level [73
The induction of angiogenesis has been described by various research groups. In 2008, a promotion of angiogenesis could be seen in exosomes secreted by glioblastoma cells [32
]. In the same disease, Svensson et al. noted activation of PAR-2 also in vascular endothelial cells. They have arisen through induced angiogenesis after hypoxic treatment [74
]. Later, mRNA of exosomes demonstrated proliferation of vascular endothelial cells [75
]. An impact while studying renal cancer cell line was seen as CD105-positive exosomes led after affection of vascular endothelial cells to a promotion of growth. CD105-negative cells maintained no effect [76
]. Subsequently, promotion of angiogenesis was shown in breast cancer, as miR-210 contained by exosomes seemed to have an impact on it and reversion by suppression of exosomes secretion stated the contrary [77
]. Similar findings were reported by Li et al. in liver cancer cells [78
The migration and invasion of cancer cells are subsequent important factors in metastasis. In colon carcinoma, a transfer of TEX induced a rise of cell proliferation and chemoresistance [79
]. In melanoma cells of patients with advanced stages of carcinoma, bone marrow-derived cells were stimulated by exosomes forming a metastatic niche and leading to a prometastatic phenotype [30
]. Also, HSP90alpha, as an exosomal surface marker, is able to activate plasminogen to lead to enhanced invasive capacity of tumor cells [80
Exosomes were studied in relation to T- and NK-cells, as well as macrophages. Exosomes possess the ability of downregulating CD69 expression on CD4+
T-cells and interfere with T-cell activation. Exosomes of cancer patients showed even higher immune suppression by decreased levels of CD69 expression. [34
] These findings confirm results of other groups [11
]. It is suspected that mRNA expression and the translation into inhibitory proteins gets enhanced by TEX.
When looking at CD8+
T-cells, coincubation with TEX lets them undergo apoptosis [11
TEX also exert effects on Treg-cells. The adenosine pathway is used by Treg-cells to operate suppression. A downregulation of mRNA coding for this pathway genes was shown in Treg-cells after coincubation with TEX. Also, TEX appear to activate resting Treg-cells through the increase of CD39 as well as CD73 production. Furthermore, calcium signalling is important for T-cell activation and T-cell-dependent immune responses. Incubation with TEX showed a calcium influx in T-cells, which was stronger than the response induced by dendritic cells [11
The contribution of B-cells was, to date, not well respected. Results of our experiments could show a suppression of activated B-cells by TEX, which seems to be another way TEX may exert protumour effects (unpublished results).
The exact mechanism of immunomodulation through TEX is still unclear. An important part seems to be the uptake of small noncoding RNA (microRNA, miRNA), which leads to alteration of RNA expression and reprogramming of recipient cells [38
]. This was, in particular, shown in immune cells, which take up exosomes as B-cells, monocytes and dendritic cells [82
]. miRNA leads to destabilisation of RMA after binding and consecutive reduction of expression of the encoded protein [84
]. Some of exosomes-mediated post-transcriptional alternations are associated with horizontal transfer of miRNA and oncogenic proteins from tumour cells to endothelial cells.
Exosomes having miRNA as their cargo are able to shape immune responses and seem responsible for protumorigenic functions in the recipient cells.
TEX signalling happens through receptor binding either on the vesicle surface or an internalisation by the recipient cell takes place. Our work could show that a direct surface interaction of exosomes with T-cells is sufficient to transfer signals. There is no internalisation needed as seen in phagocytic cells [11
]. Internalisation and binding of TEX by B-cells and monocytes appeared to be time-dependent, dose-dependent and recipient cell type dependent.
Taken together, TEX take effect in antitumor immune functions and promotion of tumour progression leading to a gain or loss of function in recipient cells and being an option used by tumours as immune escape [30
]. TEX play a dual part, having proinflammatory as well as anti-inflammatory properties. Exosomes of healthy donors did not induce immunosuppression [86
5.2. Loaded Exosomes as Targeted Drug Delivery
Yang et al. showed that unmodified exosomes are able to cross the blood–brain barrier to some extent [87
]. Brain exposure can, however, be increased by using certain brain-targeting ligands [88
Exosomes can be modified using various techniques after isolation (exogenous loading): freeze–thaw cycles, incubation, sonication, electroporation and extrusion.
On the contrary, modification can also be made during the formation of exosomes (endogenous loading).
As a breakthrough, Batrakova managed to load exosomes exogenous with the chemotherapeutic agent paclitaxel as the drug-delivery technique. Therapy was, with respect to lung metastasis within mice and in vitro, a lot more successful, having effect even on multiresistant cells and reducing required medication 50 times [90
]. Loading drugs into exosomes seems a powerful way of delivering anticancer drugs, but it may lead to increased toxicity, which has to be evaluated carefully.
Another study group loaded glioblastoma-derived exosomes with curcumin (an anti-inflammatory drug) and administered it intranasal to animals with encephalitis. It led to a clinical amelioration with induction of immune tolerance and apoptosis of activated immune cells [91
Several groups studied the delivery of small interfering RNA (siRNA) into cells using exosomes loaded with electroporation. Some report functional siRNA, others were unsuccessful in proceeding with electroporation [89
The endogenous approach uses direct transfection of RNA in interest cells, from which exosomes are later derived. Loading with miRNA and siRNA was possible and functional delivery was shown by various groups [94
]. At the moment, limiting factors of this method are unknown alterations made to the exosome donor cell due to transfection leading to the changed cargo of exosomes. Besides, when conducting transfection, the produced side-products are copurified during isolation, features of exosomes can be changed leading to false-positive effects.
These are promising results for using exosomes as drug delivery vehicles and could be the solution to therapy-resistant cells as well as tumour immune escape.
A huge advantage, the possibility of exosomes to cross the blood–brain barrier, has to be highlighted; this leads to a higher bioavailability regarding the therapeutic use of exosomes if drugs of biologics are administered to them. Additionally, the toxicological effect is selective and targeted, reducing systemic effects compared to conventional free drugs.
However, research is in the fledgling stages and loading of the vesicles can be challenging, with probable alteration of exosomal membrane and function. Another known limitation is well known in cancer research, that once the successful research moves on from animal models to humans, the observed effects may be fading.
5.4. Engineered/Designer Exosomes
The first approaches for delivery drug technologies in the context of loading already existing exosomes have already been described above. This technology bears some problems like probable missing tissue specificity and immunogenic potential.
Alvarez-Erviti et al. engineered exosomes with self-derived immature dendritic cells to reduce immunogenicity and conducted them to express Lamp2b to realise tissue-specificity. Purified exosomes were loaded with siRNA by electroporation fused to a peptide important in the treatment of Alzheimer’s disease and injected intravenously. Results showed specific delivery to the brain with a knockdown of mRNA and peptid by 60% [89
Rashid et al. highlights further engineering of exosomes and their accumulated future hopes and problems. The perfect cell used to engineer exosomes should be nonimmunogenic, have an unlimited cell passage capacity without mutating, have an ability to produce itself abundantly and be easily modified. They believe to have found these potentials in HEK293-Tcells. Mice with metastatic breast cancer showed slower tumor growth after injection with engineered exosomes combined with a better survival compared to control.
Based on HEK293-Tcells, Kojima et al. even took it a step further and designed exosomes completely out of single components. Therapeutic catalase mRNA as cargo in exosomes showed attenuated neuroinflammation in Parkinson’s disease as a positive results [101
Designer exosomes are still in their fledgling stages but deliver promising results for further research as drug-delivering technologies.
5.5. Exosomes in Immune-Modulatory Therapies
Therapy in HNSCC has been mainly limited to surgery and radio-chemotherapy in the past few years. With growing understanding of the immune system and discovery of CTLA-4, PD-1, its inhibiting functions and suitable antibodies through James Allison and Tasuku Honjo check-point inhibition was found. Antibodies of CTLA-4 showed auspicious results in metastasised melanoma. PD-1 was further studied in HNSCC.
PD-L1 is found on the surface of many cell types being a membrane-bound ligand and upregulated in inflammation or malign situations. After binding to the PD-1 receptor on immune T-cells an activation of T-cells gets suppressed important to keep inflammatory responses regulated, being a checkpoint inhibitor. Tumor cells can adapt the mechanism to evade immune destruction [102
]. After the discovery of Honjo, an immune-modulatory treatment with anti-PD-1 treatment was developed, showing some response in metastasised HNSCC. To date, it is the only FDA-approved immune-modulatory therapy with successful results.
One of the great drawbacks remains the poor response rate of 10–30% with anti-PD-1 treatment [103
]. In 2017, isolated exosomes of patients with HNSCC could be shown to carry PD-L1 and PD-1 [36
]. PD-L1-carrying exosomes correlate with clinical stage, disease activity and prognosis. Higher numbers of PD-L1positive
exosomes leads to poorer prognosis. In contrast, PD-1-containing exosomes did not match with the clinicopathological profile.
Plasma-derived exosomes in HNSCC showed biologically active PD-L1, which managed T-cell dysfunction. Anti-PD-1 was able to reverse this suppression. Maybe exosomal PD-L1, but not PD-1, is clinically relevant, possibly explaining reduced response rate with anti-PD-1 treatment and stratifying clinical responders from nonresponders. [104
]. Besides, the membrane-bound form PD-L1 was also shown to be freely soluble in blood and other liquids (sPD-L1). Comparing exosome-bound PD-L1 and sPD-L1 levels in HNSCC, the soluble form did not correlate with clinicopathological findings. Results of other studies showed alternating results [106
Exosomal PD-L1 recently moved into the spotlight as a possible therapeutic target, as suppression induces antitumour immunity and memory. Membrane-bound PD-L1 leads to diminished activity of T-cells in lymph nodes. Blocking of exosomal PD-L1 leads to a reversion and suppression of growth of local tumour cells.
Summing up, exosomal PD-L1 is an important regulator of tumour progression with the ability to suppress T-cell activation. Inhibition can lead to antitumour immunity [108