Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties. MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function. This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

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Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies

The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates EVs' capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L-EVs, 100-800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV-subtype that are distinct from small EVs (S-EVs, 30-150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density-gradient purified L-EVs (1.07-1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L-EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell-cell and cell-ECM interactions); and (ii) membrane surface-associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand-receptor analysis of L-EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L-EV proteome revealed enrichment for proteins belonging to COPI/II-coated ER/Golgi-derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L-EVs and S-EVs, our data reveals surfaceome heterogeneity between the two EV-subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L-EVs and molecular leads for future studies seeking to decipher L-EV heterogeneity and function.

Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform.

The tumor microenvironment (TME) includes a network of cancerous and non-cancerous cells, together with associated blood vessels, the extracellular matrix, and signaling molecules. The TME contributes to cancer progression during various phases of tumorigenesis, and interactions that take place within the TME have become targets of focus in cancer therapy development. Extracellular vesicles (EVs) are known to be conveyors of genetic material, proteins, and lipids within the TME. One of the hallmarks of cancer is its ability to reprogram metabolism to sustain cell growth and proliferation in a stringent environment. In this review, we provide an overview of TME EV involvement in the metabolic reprogramming of cancer and stromal cells, which favors cancer progression by enhancing angiogenesis, proliferation, metastasis, treatment resistance, and immunoevasion. Targeting the communication mechanisms and systems utilized by TME-EVs is opening a new frontier in cancer therapy.

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The Role of Extracellular Vesicles in Metabolic Reprogramming of the Tumor Microenvironment

Pancreatic cancer (PC) is a devastating disease, offering poor mortality rates for patients. The current challenge being faced is the inability to diagnose patients in a timely manner, where potentially curative resection provides the best chance of survival. Recently, small/nanosized extracellular vesicles (sEVs), including exosomes, have gained significant preclinical and clinical attention due to their emerging roles in cancer progression and diagnosis. Extracellular vesicles (EVs) possess endogenous properties that offer stability and facilitate crossing of biological barriers for delivery of molecular cargo to cells, acting as a form of intercellular communication to regulate function and phenotype of recipient cells. This review provides an overview of the role of EVs, their subtypes and their oncogenic cargo (as characterised by targeted studies as well as agnostic '-omics' analyses) in the pathobiology of pancreatic cancer. The discussion covers the progress of 'omics technology' that has enabled elucidation of the molecular mechanisms that mediate the role of EVs and their cargo in pancreatic cancer progression. 

Small extracellular vesicles (exosomes) and their cargo in pancreatic cancer: Key roles in the hallmarks of cancer.

In recent years, tremendous progress has been made in understanding the roles of extracellular vesicles (EVs) in cancer. Thanks to advancements in molecular biology, it has been found that the fraction of EVs called exosomes or small EVs (sEVs) modulates the sensitivity of cancer cells to chemotherapeutic agents by delivering molecularly active non-coding RNAs (ncRNAs). An in-depth analysis shows that two main molecular mechanisms are involved in exosomal modified chemoresistance: (1) translational repression of anti-oncogenes by exosomal microRNAs (miRs) and (2) lack of translational repression of oncogenes by sponging of miRs through long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). At the cellular level, these processes increase the proliferation and survival of cancer cells and improve their ability to metastasize and resist apoptosis. In addition, studies in animal models have shown enhancing tumor size under the influence of exosomal ncRNAs. Ultimately, exosomal ncRNAs are responsible for clinically significant chemotherapy failures in patients with different types of cancer. Preliminary data have also revealed that exosomal ncRNAs can overcome chemotherapeutic agent resistance, but the results are thoroughly fragmented. This review presents how exosomes modulate the response of cancer cells to chemotherapeutic agents. Understanding how exosomes interfere with chemoresistance may become a milestone in developing new therapeutic options, but more data are still required.

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Small Extracellular Vesicles and Their Involvement in Cancer Resistance: An Up-to-Date Review

Extracellular vesicles (EVs) hold great promise as therapeutic modalities due to their endogenous characteristics, however, further bioengineering refinement is required to address clinical and commercial limitations. Clinical applications of EV-based therapeutics are being trialed in immunomodulation, tissue regeneration and recovery, and as delivery vectors for combination therapies. Native/biological EVs possess diverse endogenous properties that offer stability and facilitate crossing of biological barriers for delivery of molecular cargo to cells, acting as a form of intercellular communication to regulate function and phenotype. Moreover, EVs are important components of paracrine signaling in stem/progenitor cell-based therapies, are employed as standalone therapies, and can be used as a drug delivery system. Despite remarkable utility of native/biological EVs, they can be improved using bio/engineering approaches to further therapeutic potential. EVs can be engineered to harbor specific pharmaceutical content, enhance their stability, and modify surface epitopes for improved tropism and targeting to cells and tissues in vivo. Limitations currently challenging the full realization of their therapeutic utility include scalability and standardization of generation, molecular characterization for design and regulation, therapeutic potency assessment, and targeted delivery. The fields' utilization of advanced technologies (imaging, quantitative analyses, multi-omics, labeling/live-cell reporters), and utility of biocompatible natural sources for producing EVs (plants, bacteria, milk) will play an important role in overcoming these limitations. Advancements in EV engineering methodologies and design will facilitate the development of EV-based therapeutics, revolutionizing the current pharmaceutical landscape.

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Development of Extracellular Vesicle Therapeutics: Challenges, Considerations, and Opportunities.

Extracellular vesicles (EVs) from stem cells have shown significant therapeutic potential to repair injured cardiac tissues and regulate pathological fibrosis. However, scalable generation of stem cells and derived EVs for clinical utility remains a huge technical challenge. Here, we report a rapid size-based extrusion strategy to generate EV-like membranous nanovesicles (NVs) from easily sourced human iPSCs in large quantities (yield 900× natural EVs). NVs isolated using density-gradient separation (buoyant density 1.13 g/mL) are spherical in shape and morphologically intact and readily internalised by human cardiomyocytes, primary cardiac fibroblasts, and endothelial cells. NVs captured the dynamic proteome of parental cells and include pluripotency markers (LIN28A, OCT4) and regulators of cardiac repair processes, including tissue repair (GJA1, HSP20/27/70, HMGB1), wound healing (FLNA, MYH9, ACTC1, ILK), stress response/translation initiation (eIF2S1/S2/S3/B4), hypoxia response (HMOX2, HSP90, GNB1), and extracellular matrix organization (ITGA6, MFGE8, ITGB1). Functionally, NVs significantly promoted tubule formation of endothelial cells (angiogenesis) (p < 0.05) and survival of cardiomyocytes exposed to low oxygen conditions (hypoxia) (p < 0.0001), as well as attenuated TGF-β mediated activation of cardiac fibroblasts (p < 0.0001). Quantitative proteome profiling of target cell proteome following NV treatments revealed upregulation of angiogenic proteins (MFGE8, MYH10, VDAC2) in endothelial cells and pro-survival proteins (CNN2, THBS1, IGF2R) in cardiomyocytes. In contrast, NVs attenuated TGF-β-driven extracellular matrix remodelling capacity in cardiac fibroblasts (ACTN1, COL1A1/2/4A2/12A1, ITGA1/11, THBS1). This study presents a scalable approach to generating functional NVs for cardiac repair.

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Jonathan Lozano

Papers

Development of Extracellular Vesicle Therapeutics: Challenges, Considerations, and Opportunities.

Scalable Generation of Nanovesicles from Human-Induced Pluripotent Stem Cells for Cardiac Repair