Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting.

Haematopoiesis dysregulation with the presence of immature myeloid and erythroid immunosuppressive cells are key characteristics of the immune escape phase of tumour development. Here, the role of in vitro generated B16F10 tumour cell-derived extracellular vesicles (tEVs) as indirect cellular communicators, participating in tumour-induced dysregulation of haematopoiesis, was explored. The isolated tEVs displayed features of small EVs with a size range of 100-200 nm, expressed the common EV markers CD63, CD9, and Alix, and had a spherical shape with a lipid bilayer membrane. Proteomic profiling revealed significant levels of angiogenic factors, particularly vascular endothelial growth factor (VEGF), osteopontin, and tissue factor, associated with the tEVs. Systemic administration of these tEVs in syngeneic mice induced splenomegaly and disrupted haematopoiesis, leading to extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and the development of anaemia. These effects closely mirrored those observed in tumour-bearing mice and were not seen after heat inactivating the tEVs. In vitro studies demonstrated that tEVs independently induced the expansion of bone marrow granulocytic myeloid suppressor cells and B cells while reducing the frequency of cells in the erythropoietic lineage. These effects of tEVs were significantly abrogated by the blockade of VEGF or heat inactivation. Our findings underscore the important role of tEVs in dysregulating haematopoiesis during the immune escape phase of cancer immunoediting, suggesting their potential as targets for addressing immune evasion and reinstating normal hematopoietic processes.

Antibody-displaying extracellular vesicles for targeted cancer therapy.

Extracellular vesicles (EVs) function as natural delivery vectors and mediators of biological signals across tissues. Here, by leveraging these functionalities, we show that EVs decorated with an antibody-binding moiety specific for the fragment crystallizable (Fc) domain can be used as a modular delivery system for targeted cancer therapy. The Fc-EVs can be decorated with different types of immunoglobulin G antibody and thus be targeted to virtually any tissue of interest. Following optimization of the engineered EVs by screening Fc-binding and EV-sorting moieties, we show the targeting of EVs to cancer cells displaying the human epidermal receptor 2 or the programmed-death ligand 1, as well as lower tumour burden and extended survival of mice with subcutaneous melanoma tumours when systemically injected with EVs displaying an antibody for the programmed-death ligand 1 and loaded with the chemotherapeutic doxorubicin. EVs with Fc-binding domains may be adapted to display other Fc-fused proteins, bispecific antibodies and antibody-drug conjugates.

Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

The immune escape stage in cancer immunoediting is a pivotal feature, transitioning immune-controlled tumor dormancy to progression, and augmenting invasion and metastasis. Tumors employ diverse mechanisms for immune escape, with generating immunosuppressive cells from skewed hematopoiesis being a crucial mechanism. This led us to suggest that tumor cells with immune escape properties produce factors that induce dysregulations in hematopoiesis. In support of this suggestion, this study found that mice bearing advanced-stage tumors exhibited dysregulated hematopoiesis characterized by the development of splenomegaly, anemia, extramedullary hematopoiesis, production of immunosuppressive mediators, and expanded medullary myelopoiesis. Further ex vivo studies exhibited that conditioned medium derived from EL4lu2 cells could mediate the expansion of myeloid derived suppressor cells (MDSCs) in bone marrow cell cultures. The protein array profiling results revealed the presence of elevated levels of osteopontin (OPN), prostaglandin E2 (PGE2) and interleukin 17 (IL-17) in the culture medium derived from EL4luc2 cells. Accordingly, substantial levels of these factors were also detected in the sera of mice bearing EL4luc2 tumors. Among these factors, only PGE2 alone could increase the number of MDSCs in the BM cell cultures. This effect of PGE2 was significantly potentiated by the presence of OPN but not IL-17. Finally, in vitro treatment of EL4luc2 cells with pioglitazone, a modulator of OPN and cyclooxygenase 2 (COX-2) resulted in a significant reduction in cell proliferation in EL4luc2 cells. Our findings highlight the significant role played by tumor cell-derived OPN and PGE2 in fostering the expansion of medullary MDSCs and in promoting tumor cell proliferation. Furthermore, these intertwined cancer processes could be key targets for pioglitazone intervention.

Insights into cellular behavior and micromolecular communication in urothelial micrografts.

Autologous micrografting is a technique currently applied within skin wound healing, however, the potential use for surgical correction of other organs with epithelial lining, including the urinary bladder, remains largely unexplored. Currently, little is known about the micrograft expansion potential and the micromolecular events that occur in micrografted urothelial cells. In this study, we aimed to evaluate the proliferative potential of different porcine urothelial micrograft sizes in vitro, and, furthermore, to explore how urothelial micrografts communicate and which microcellular events are triggered. We demonstrated that increased tissue fragmentation subsequently potentiated the yield of proliferative cells and the cellular expansion potential, which confirms, that the micrografting principles of skin epithelium also apply to uroepithelium. Furthermore, we targeted the expression of the extracellular signal-regulated kinase (ERK) pathway and demonstrated that ERK activation occurred predominately at the micrograft borders and that ERK inhibition led to decreased urothelial migration and proliferation. Finally, we successfully isolated extracellular vesicles from the micrograft culture medium and evaluated their contents and relevance within various enriched biological processes. Our findings substantiate the potential of applying urothelial micrografting in future tissue-engineering models for reconstructive urological surgery, and, furthermore, highlights certain mechanisms as potential targets for future wound healing treatments.

Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles.

Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a robust cargo loading strategy. To address the paucity of such scaffold proteins, we leverage a simple and reliable assay that can distinguish intravesicular cargo proteins from surface- as well as non-vesicular proteins and compare the EV-sorting potential of 244 candidate proteins. We identify 24 proteins with conserved EV-sorting abilities across five types of producer cells. TSPAN2 and TSPAN3 emerge as lead candidates and outperform the well-studied CD63 scaffold. Importantly, these engineered EVs show promise as delivery vehicles in cell cultures and mice as demonstrated by efficient transfer of luminal cargo proteins as well as surface display of different functional entities. The discovery of these scaffolds provides a platform for EV-based engineering.

Extracellular vesicles engineered to bind albumin demonstrate extended circulation time and lymph node accumulation in mouse models.

Extracellular vesicles (EVs) have shown promise as potential therapeutics for the treatment of various diseases. However, their rapid clearance after administration could be a limitation in certain therapeutic settings. To solve this, an engineering strategy is employed to decorate albumin onto the surface of the EVs through surface display of albumin binding domains (ABDs). ABDs were either included in the extracellular loops of select EV-enriched tetraspanins (CD63, CD9 and CD81) or directly fused to the extracellular terminal of single transmembrane EV-sorting domains, such as Lamp2B. These engineered EVs exert robust binding capacity to human serum albumins (HSA) in vitro and mouse serum albumins (MSA) after injection in mice. By binding to MSA, circulating time of EVs dramatically increases after different routes of injection in different strains of mice. Moreover, these engineered EVs show considerable lymph node (LN) and solid tumour accumulation, which can be utilized when using EVs for immunomodulation, cancer- and/or immunotherapy. The increased circulation time of EVs may also be important when combined with tissue-specific targeting ligands and could provide significant benefit for their therapeutic use in a variety of disease indications.

Identification of storage conditions stabilizing extracellular vesicles preparations.

Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS-HAT). We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80°C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

Growth Media Conditions Influence the Secretion Route and Release Levels of Engineered Extracellular Vesicles.

Extracellular vesicles (EVs) are nanosized cell-derived vesicles produced by all cells, which provide a route of intercellular communication by transmitting biological cargo. While EVs offer promise as therapeutic agents, the molecular mechanisms of EV biogenesis are not yet fully elucidated, in part due to the concurrence of numerous interwoven pathways which give rise to heterogenous EV populations in vitro. The equilibrium between the EV-producing pathways is heavily influenced by factors in the extracellular environment, in such a way that can be taken advantage of to boost production of engineered EVs. In this study, a quantifiable EV-engineering approach is used to investigate how different cell media conditions alter EV production. The presence of serum, exogenous EVs, and other signaling factors in cell media alters EV production at the physical, molecular, and transcriptional levels. Further, it is demonstrated that the ceramide-dependent EV biogenesis route is the major pathway to production of engineered EVs during optimized EV-production. These findings suggest a novel understanding to the mechanisms underlying EV production in cell culture which can be applied to develop advanced EV production methods.

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles.

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

Novel Orthogonally Hydrocarbon-Modified Cell-Penetrating Peptide Nanoparticles Mediate Efficient Delivery of Splice-Switching Antisense Oligonucleotides In Vitro and In Vivo.

Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features.

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

Optimised Electroporation for Loading of Extracellular Vesicles with Doxorubicin.

The clinical use of chemotherapeutics is limited by several factors, including low cellular uptake, short circulation time, and severe adverse effects. Extracellular vesicles (EVs) have been suggested as a drug delivery platform with the potential to overcome these limitations. EVs are cell-derived, lipid bilayer nanoparticles, important for intercellular communication. They can transport bioactive cargo throughout the body, surmount biological barriers, and target a variety of tissues. Several small molecule drugs have been successfully incorporated into the lumen of EVs, permitting efficient transport to tumour tissue, increasing therapeutic potency, and reducing adverse effects. However, the cargo loading is often inadequate and refined methods are a prerequisite for successful utilisation of the platform. By systematically evaluating the effect of altered loading parameters for electroporation, such as total number of EVs, drug to EV ratio, buffers, pulse capacitance, and field strength, we were able to distinguish tendencies and correlations. This allowed us to design an optimised electroporation protocol for loading EVs with the chemotherapeutic drug doxorubicin. The loading technique demonstrated improved cargo loading and EV recovery, as well as drug potency, with a 190-fold increased response compared to naked doxorubicin.

Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging.

Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or real-time imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration.