In the era of the rapid development of cancer immunotherapy, there is a high level of interest in the application of cell-released small vesicles that stimulate the immune system. As cell-derived nanovesicles, exosomes show great promise in cancer immunotherapy because of their immunogenicity and molecular transfer function. The cargoes carried on exosomes have been recently identified with improved technological advances and play functional roles in the regulation of immune responses. In particular, exosomes derived from tumor cells and immune cells exhibit unique composition profiles that are directly involved in anticancer immunotherapy. More importantly, exosomes can deliver their cargoes to targeted cells and thus influence the phenotype and immune-regulation functions of targeted cells. Accumulating evidence over the last decade has further revealed that exosomes can participate in multiple cellular processes contributing to cancer development and therapeutic effects, showing the dual characteristics of promoting and suppressing cancer. The potential of exosomes in the field of cancer immunotherapy is huge, and exosomes may become the most effective cancer vaccines, as well as targeted antigen/drug carriers. Understanding how exosomes can be utilized in immune therapy is important for controlling cancer progression; additionally, exosomes have implications for diagnostics and the development of novel therapeutic strategies. This review discusses the role of exosomes in immunotherapy as carriers to stimulate an anti-cancer immune response and as predictive markers for immune activation; furthermore, it summarizes the mechanism and clinical application prospects of exosome-based immunotherapy in human cancer.

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Exosome-based immunotherapy: a promising approach for cancer treatment

Efficient targeted delivery of anticancer agents to TNBC cells remains one of the greatest challenges to developing therapies. The lack of tumor-specific markers, aggressive nature of the tumor, and unique propensity to recur and metastasize make TNBC tumors more difficult to treat than other subtypes. We propose to exploit natural ability of macrophages to target cancer cells by means of extracellular vesicles (EVs) as drug delivery vehicles for chemotherapeutic agents, paclitaxel (PTX) and doxorubicin (Dox). We demonstrated earlier that macrophage-derived EVs loaded with PTX (EV-PTX) and Dox (EV-Dox) target cancer cells and exhibited high anticancer efficacy in a mouse model of pulmonary metastases. Herein, we report a manufacture and characterization of novel EV-based drug formulations using different loading procedures that were optimized by varying pH, temperature, and sonication conditions. Selected EV-based formulations showed a high drug loading, efficient accumulation in TNBC cells in vitro, and pronounced anti-proliferation effect. Drug-loaded EVs target TNBC in vivo, including the orthotopic mouse T11 tumors in immune competent BALB/C mice, and human MDA-MB-231 tumors in athymic nu/nu mice, and abolished tumor growth. Overall, EV-based formulations can provide a novel solution to a currently unmet clinical need and reduce the morbidity and mortality of TNBC patients. Graphical Abstract Macrophage-derived extracellular vesicles (EVs) for targeted drug delivery to TNBC tumors. Chemotherapeutics with different water solubility (Dox or PTX, i.e. hydrophilic or hydrophobic drugs, respectively) were loaded into macrophage-derived EVs through parental cells (Dox), or into naive EVs (Dox or PTX), and their antitumor efficacy was demonstrated in mouse orthotopic TNBC model.

Macrophage-Derived Extracellular Vesicles as Drug Delivery Systems for Triple Negative Breast Cancer (TNBC) Therapy

Chronic, subclinical inflammation was often observed in the diabetic wound area, causing inadequate and delayed wound-healing effects by failing to initiate cell migration, proliferation, and extra...

Macrophage-derived exosomes accelerate wound healing through their anti-inflammation effects in a diabetic rat model

Inflammasomes are intracellular protein complexes of pattern recognition receptors and caspase-1, with essential functions in regulating inflammatory responses of macrophages and dendritic cells. The primary role of inflammasomes is to catalyze processing and secretion of pro-inflammatory cytokines IL-1β and IL-18. Recently, intracellular non-canonical inflammasome activation by caspases-4/5, which are also regulators of pyroptosis via processing gasdermin D, has been elucidated. Caspase-1, the effector protease of inflammasome complex, is also known to modulate secretion of large number of other proteins. Thereby, besides its known role in processing pro-inflammatory cytokines, the inflammasome turns into a universal regulator of protein secretion, which allows the danger-exposed cells to release various proteins in order to alert and guide neighboring cells. Majority of these proteins are not secreted through the conventional ER-Golgi secretory pathway. Instead, they are segregated in membrane-enclosed compartment and secreted in nanosized extracellular vesicles, which protect their cargo and guide it for delivery. Growing evidence indicates that inflammasome activity correlates with enhanced secretion of extracellular vesicles and modulation of their protein cargo. This inflammasome-driven unconventional, vesicle-mediated secretion of multitude of immunoregulatory proteins may constitute a novel paradigm in inflammatory responses. In this mini review we discuss the current knowledge and highlight unsolved questions about metabolic processes, signals, and mechanisms linking inflammasome activity with regulated extracellular vesicle secretion of proteins. Further investigations on this relationship may in the future help understanding the significance of extracellular vesicle secretion in inflammatory diseases such as atherosclerosis, gouty arthritis, asthma, Alzheimer’s and many others.

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From Inflammasome to Exosome-Does Extracellular Vesicle Secretion Constitute an Inflammasome-Dependent Immune Response?

Exosome is a nanoscale vesicle with a size range of 30-100 nm. It is secreted from cell to extracellular space by exocytosis after fusion of multivesicular body (MVB) (formed by endocytic vesicles) with plasma membrane. Exosome plays several important roles in cellular homeostasis and intercellular communications. During the last two decades, exosome has acquired a wide attention to explore its additional roles in various aspects of cell biology and function in several organ systems. For the kidney, several lines of evidence have demonstrated 1that exosome is involved in the renal physiology and pathogenic mechanisms of various kidney diseases/disorders. This article summarizes roles of the exosome as the potential source of biomarkers, pathogenic molecules, and therapeutic biologics that have been extensively investigated in many kidney diseases/disorders, including lupus nephritis (LN), other glomerular diseases, acute kidney injury (AKI), diabetic nephropathy (DN), as well as in the process of renal fibrosis and chronic kidney disease (CKD) progression, in addition to polycystic kidney disease (PKD), kidney transplantation, and renal cell carcinoma (RCC). Moreover, the most recent evidence has shown its emerging role in kidney stone disease (or nephrolithiasis), involving inflammasome activation and inflammatory cascade frequently found in kidney stone pathogenesis.

Roles for Exosome in Various Kidney Diseases and Disorders.

In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) after exposure to calcium oxalate monohydrate (COM) crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoLC-ESI-ETD MS/MS as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ) regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90β) and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1β (IL-1β), one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of pro-inflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA (siRNA) targeting on vimentin (si-Vimentin). Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals.

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Roles of Macrophage Exosomes in Immune Response to Calcium Oxalate Monohydrate Crystals.

Several lines of evidence have demonstrated anti-fibrotic property of epigallocatechin-3-gallate (EGCG) in many tissues/organs but with unclear mechanisms. This study thus aimed to define cellular mechanisms underlying such protective effect of EGCG. HK-2 renal cells were treated with 5 ng/ml TGF-β1 for 24 h with/without pretreatment by 5 μM EGCG for 1 h. The cells were then evaluated by morphological examination, immunofluorescence study, semi-quantitative RT-PCR, Western blotting, and atomic force microscopy (AFM). The results showed that TGF-β1-treated cells underwent epithelial mesenchymal transition (EMT) as evidenced by morphological change into fibroblast-like and increases in spindle index, mesenchymal markers (Snail1 and vimentin), extracellular matrix (fibronectin), cell stiffness (by AFM measurement) and actin stress fibers, whereas the epithelial markers (E-cadherin and ZO-1) were decreased. All of these features were abolished by EGCG pretreatment. Functional studies revealed that the anti-fibrotic property of EGCG was, at least in part, due to de-activation/stabilization of GSK-3β/β-catenin/Snail1 (EMT-triggering) signaling pathway that was activated by TGF-β1 as shown by maintaining phosphorylated GSK-3β, β-catenin and Snail1 to their basal levels. Additionally, Nrf2 knockdown by small interfering RNA could abolish the EGCG effect on β-catenin expression. These data indicate that EGCG attenuates TGF-β1-induced EMT in renal tubular cells through GSK-3β/β-catenin/Snail1 and Nrf2 pathways.

Epigallocatechin-3-gallate prevents TGF-β1-induced epithelial-mesenchymal transition and fibrotic changes of renal cells via GSK-3β/β-catenin/Snail1 and Nrf2 pathways

Caffeine has been demonstrated to possess anti-fibrotic activity against liver fibrosis. However, its role in renal fibrosis remained unclear. This study investigated the effects of caffeine on renal fibroblast activation induced by hypoxia (one of the inducers for renal fibrosis). BHK-21 fibroblasts were cultured under normoxia or hypoxia with or without caffeine treatment. Hypoxia increased levels of fibronectin, α-smooth muscle actin, actin stress fibers, intracellular reactive oxygen species (ROS), and oxidized proteins. However, caffeine successfully preserved all these activated fibroblast markers to their basal levels. Cellular catalase activity was dropped under hypoxic condition but could be reactivated by caffeine. Hif1a gene and stress-responsive Nrf2 signaling molecule were elevated/activated by hypoxia, but only Nrf2 could be partially recovered by caffeine. These data suggest that caffeine exhibits anti-fibrotic effect against hypoxia-induced renal fibroblast activation through its antioxidant property to eliminate intracellular ROS, at least in part, via downstream catalase and Nrf2 mechanisms.

https://tandfonline.com/doi/pdf/10.1080/19336918.2019.1638691

Angkhana Nilnumkhum

Papers

Roles of Macrophage Exosomes in Immune Response to Calcium Oxalate Monohydrate Crystals.

Epigallocatechin-3-gallate prevents TGF-β1-induced epithelial-mesenchymal transition and fibrotic changes of renal cells via GSK-3β/β-catenin/Snail1 and Nrf2 pathways

Caffeine inhibits hypoxia-induced renal fibroblast activation by antioxidant mechanism.