Landscape of extracellular vesicles in the tumour microenvironment: Interactions with stromal cells and with non-cell components, and impacts on metabolic reprogramming, horizontal transfer of neoplastic traits, and the emergence of therapeutic resistance.

Extracellular vesicles (EVs) are increasingly recognised as a pivotal player in cell-cell communication, an attribute of EVs that derives from their ability to transport bioactive cargoes between cells, resulting in complex intercellular signalling mediated by EVs, which occurs under both physiological and pathological conditions. In the context of cancer, recent studies have demonstrated the versatile and crucial roles of EVs in the tumour microenvironment (TME). Here, we revisit EV biology, and focus on EV-mediated interactions between cancer cells and stromal cells, including fibroblasts, immune cells, endothelial cells and neurons. In addition, we focus on recent reports indicating interactions between EVs and non-cell constituents within the TME, including the extracellular matrix. We also review and summarise the intricate cancer-associated network modulated by EVs, which promotes metabolic reprogramming, horizontal transfer of neoplastic traits, and therapeutic resistance in the TME. We aim to provide a comprehensive and updated landscape of EVs in the TME, focusing on oncogenesis, cancer progression and therapeutic resistance, together with our future perspectives on the field.

Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious and poorly understood disease. To understand immune dysregulation in ME/CFS, we use single-cell RNA sequencing (scRNA-seq) to examine immune cells in patient and control cohorts. Postexertional malaise (PEM), an exacerbation of symptoms following strenuous exercise, is a characteristic symptom of ME/CFS. To detect changes coincident with PEM, we applied scRNA-seq on the same cohorts following exercise. At baseline, ME/CFS patients display classical monocyte dysregulation suggestive of inappropriate differentiation and migration to tissue. We identify both diseased and more normal monocytes within patients, and the fraction of diseased cells correlates with disease severity. Comparing the transcriptome at baseline and postexercise challenge, we discover patterns indicative of improper platelet activation in patients, with minimal changes elsewhere in the immune system. Taken together, these data identify immunological defects present at baseline in patients and an additional layer of dysregulation in platelets.

Red Blood Cell-Derived Extracellular Vesicles Display Endogenous Antiviral Effects and Enhance the Efficacy of Antiviral Oligonucleotide Therapy.

The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that unmodified red blood cell-derived extracellular vesicles (RBCEVs) can inhibit SARS-CoV-2 infection in a phosphatidylserine (PS) dependent manner. Using T cell immunoglobulin mucin domain-1 (TIM-1) as an example, we demonstrate that PS receptors on cells can significantly increase the adsorption and infection of authentic and pseudotyped SARS-CoV-2 viruses. RBCEVs competitively inhibit this interaction and block TIM-1-mediated viral entry into cells. We further extend the therapeutic efficacy of this antiviral treatment by loading antisense oligonucleotides (ASOs) designed to target conserved regions of key SARS-CoV-2 genes into RBCEVs. We establish that ASO-loaded RBCEVs are efficiently taken up by cells in vitro and in vivo to suppress SARS-CoV-2 replication. Our findings indicate that this RBCEV-based SARS-CoV-2 therapeutic displays promise as a potential treatment capable of inhibiting SARS-CoV-2 entry and replication.

Robust delivery of RIG-I agonists using extracellular vesicles for anti-cancer immunotherapy.

The RIG-I pathway can be activated by RNA containing 5' triphosphate, leading to type I interferon release and immune activation. Hence, RIG-I agonists have been used to induce immune responses against cancer as potential immunotherapy. However, delivery of 5' triphosphorylated RNA molecules as RIG-I agonists to tumour cells in vivo is challenging due to the susceptibility of these molecules to degradation. In this study, we demonstrate the use of extracellular vesicles (EVs) from red blood cells (RBCs), which are highly amenable for RNA loading and taken up robustly by cancer cells, for RIG-I agonist delivery. We evaluate the anti-cancer activity of two novel RIG-I agonists, the immunomodulatory RNA (immRNA) with a unique secondary structure for efficient RIG-I activation, and a 5' triphosphorylated antisense oligonucleotide with dual function of RIG-I activation and miR-125b inhibition (3p-125b-ASO). We find that RBCEV-delivered immRNA and 3p-125b-ASO trigger the RIG-I pathway, and induce cell death in both mouse and human breast cancer cells. Furthermore, we observe a significant suppression of tumour growth coupled with increased immune cell infiltration mediated by the activation of RIG-I cascade after multiple intratumoral injections of RBCEVs loaded with immRNA or 3p-125b-ASO. Targeted delivery of immRNA using RBCEVs with EGFR-binding nanobody administrated via intrapulmonary delivery facilitates the accumulation of RBCEVs in metastatic cancer cells, leading to potent tumour-specific CD8+ T cells immune response. This contributes to prominent suppression of breast cancer metastasis in the lung. Hence, this study provides a new strategy for efficient RIG-I agonist delivery using RBCEVs for immunotherapy against cancer and cancer metastasis.

Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy.

The advent of novel therapeutics in recent years has urged the need for a safe, non-immunogenic drug delivery vector capable of delivering therapeutic payloads specifically to diseased cells, thereby increasing therapeutic efficacy and reducing side effects. Extracellular vesicles (EVs) have garnered attention in recent years as a potentially ideal vector for drug delivery, taking into account their intrinsic ability to transfer bioactive cargo to recipient cells and their biocompatible nature. However, natural EVs are limited in their therapeutic potential and many challenges need to be overcome before engineered EVs satisfy the levels of efficiency, stability, safety and biocompatibility required for therapeutic use. Here, we demonstrate that an enzyme-mediated surface functionalization method in combination with streptavidin-mediated conjugation results in efficient surface functionalization of EVs. Surface functionalization using the above methods permits the stable and biocompatible conjugation of peptides, single domain antibodies and monoclonal antibodies at high copy number on the EV surface. Functionalized EVs demonstrated increased accumulation in target cells expressing common cancer associated markers such as CXCR4, EGFR and EpCAM both in vitro and in vivo. The functionality of this approach was further highlighted by the ability of targeting EVs to specifically deliver therapeutic antisense oligonucleotides to a metastatic breast tumor model, resulting in increased knockdown of a targeted oncogenic microRNA and improved metastasis suppression. The method was also used to equip EVs with a bifunctional peptide that targets EVs to leukemia cells and induces apoptosis, leading to leukemia suppression. Moreover, we conducted extensive testing to verify the biocompatibility, and safety of engineered EVs for therapeutic use, suggesting that surface modified EVs can be used for repeated dose treatment with no detectable adverse effects. This modular, biocompatible method of EV engineering offers a promising avenue for the targeted delivery of a range of therapeutics while addressing some of the safety concerns associated with EV-based drug delivery.

αvß1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin-3.

Breast cancer cells release a large quantity of biocargo-bearing extracellular vesicles (EVs), which mediate intercellular communication within the tumour microenvironment and promote metastasis. To identify EV-bound proteins related to metastasis, we used mass spectrometry to profile EVs from highly and poorly metastatic breast cancer lines of human and mouse origins. Comparative mass spectrometry indicated that integrins, including αv and ß1 subunits, are preferentially enriched in EVs of highly metastatic origin over those of poorly metastatic origin. These results are consistent with our histopathological findings, which show that integrin αv is associated with disease progression in breast cancer patients. Integrin αv colocalizes with the multivesicular-body marker CD63 at a higher frequency in the tumour and is enriched in circulating EVs of breast cancer patients at late stages when compared with circulating EVs from early-stage patients. With a magnetic bead-based flow cytometry assay, we confirmed that integrins αv and ß1 are enriched in the CD63+ subsets of EVs from both human and mouse highly metastatic cells. By analysing the level of integrin αv on circulating EVs, this assay could predict the metastatic potential of a xenografted mouse model. To explore the export mechanism of integrins into EVs, we performed immunoprecipitation mass spectrometry and identified members of the galectin family as potential shuttlers of integrin αvß1 into EVs. In particular, knockdown of galectin-3, but not galectin-1, causes a reduction in the levels of cell surface integrins ß1 and αv, and decreases the colocalization of these integrins with CD63. Importantly, knockdown of galectin-3 leads to a decrease of integrin αvß1 export into the EVs concomitant with a decrease in the metastatic potential of breast cancer cells. Moreover, inhibition of the integrin αvß1 complex leads to a reduction in the binding of EVs to fibronectin, suggesting that integrin αvß1 is important for EV retention in the extracellular matrix. EVs retained in the extracellular matrix are taken up by fibroblasts, which differentiate into cancer associated fibroblasts. In summary, our data indicate an important link between EV-bound integrin αvß1 with breast cancer metastasis and provide additional insights into the export of integrin αvß1 into EVs in the context of metastasis.

microRNA exchange via extracellular vesicles in cancer.

Cells utilize different means of inter-cellular communication to function properly. Here, we review the crosstalk between cancer cells and their surrounding environment through microRNA (miRNA)-containing extracellular vesicles (EVs). The current findings suggest that the export of miRNAs and uptake of miRNA-containing EVs might be an active process. As post-transcriptional regulators of gene expression, cancer-derived miRNAs that are taken up by normal cells can change the translational profile of the recipient cell towards a transformed proteome. Stromal cells can also deliver miRNAs via EVs to cancer cells to support tumour growth and cancer progression. Therefore, gaining a better understanding of EV-mediated inter-cellular communication in the tumour microenvironment might lead to the development of novel diagnostic and therapeutic strategies.

Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b.

Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts.

Efficient RNA drug delivery using red blood cell extracellular vesicles.

Most of the current methods for programmable RNA drug therapies are unsuitable for the clinic due to low uptake efficiency and high cytotoxicity. Extracellular vesicles (EVs) could solve these problems because they represent a natural mode of intercellular communication. However, current cellular sources for EV production are limited in availability and safety in terms of horizontal gene transfer. One potentially ideal source could be human red blood cells (RBCs). Group O-RBCs can be used as universal donors for large-scale EV production since they are readily available in blood banks and they are devoid of DNA. Here, we describe and validate a new strategy to generate large-scale amounts of RBC-derived EVs for the delivery of RNA drugs, including antisense oligonucleotides, Cas9 mRNA, and guide RNAs. RNA drug delivery with RBCEVs shows highly robust microRNA inhibition and CRISPR-Cas9 genome editing in both human cells and xenograft mouse models, with no observable cytotoxicity.