Exosomal Cargo Research Articles

Direct profiling of breast cancer-derived extracellular vesicles using Pd-perovskite electrochemical biosensing platform

Extracellular vesicles (EVs) harbor several signaling molecules to maintain intercellular communication. Based on the exosomal cargo type, metabolic, genomic, and proteomic status of parent cells can be investigated. Due to the existence of trivial levels of target molecules inside EVs, the application of accurate and sensitive detection methods is mandatory. Here, we used an electrochemical immunosensor using a biotinylated monoclonal CD63 antibody as the capturing element for the detection of EVs isolated from MDA-MB-231 cells and cancer patients. Simultaneously, breast cancer biomarker CA-15-3 was detected in isolated EVs using a sandwich method to increase specificity. Data indicated a linear dynamic range of 2000–10000 EVs/µL and a lower limit of quantification of 2000 EVs/µL. Based on data from real sample analysis, the levels of exosomal CA-15-3 can differ according to the severity and systemic content of this factor. Pd-perovskite-based immunosensor provides a platform for quick and in-depth analysis of EVs isolated for cancer cells.

Tipifarnib Reduces Extracellular Vesicles and Protects From Heart Failure.

Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles, including small extracellular vesicles or exosomes, and their molecular cargo are known to modulate cell-to-cell communication during multiple cardiac diseases. However, the role of systemic extracellular vesicle biogenesis inhibition in HF models is not well documented and remains unclear. We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of tipifarnib, a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a [Ras associated binding protein 27a], nSMase2 [neutral sphingomyelinase 2], and Alix [ALG-2-interacting protein X]) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery were randomly assigned to groups with and without tipifarnib treatment (10 mg/kg 3 times/wk) and monitored for 8 weeks, and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies. TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced left ventricular dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of tipifarnib in TAC mice reduced circulating exosomes to baseline and remarkably improved left ventricular functions, hypertrophy, and fibrosis. Tipifarnib treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR 331-5p, which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR 331-5p is crucial for inhibiting the fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR 331-5p that acts as a potent repressor for one of the fibrotic mechanisms mediated by HOXC8. Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib-mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling in HF.

#1978 Proteomic analysis of exosomes derived from mesangial cells treated with galactose-deficient IgA1

Abstract Background and Aims IgA nephropathy (IgAN) is an autoimmune disease characterized by IgA deposits that build up in the kidneys, inflame and damage glomeruli causing hematuria and proteinuria. It is a leading cause of chronic kidney disease, which often results in end-stage kidney disease. There is no disease specific treatment for IgAN. The glomerulus, in which IgA1 lacking galactose residues (gdIgA1) are deposited, consists of mesangial cells, podocytes, and endothelial cells. Crosstalk between the cell types allows the glomerulus to act as a unit and react to its dynamic environment. There is very little evidence on mesangial cell derived exosomes and their cargo to understand the pathological crosstalk signaling in the glomerulus of IgAN. We isolated exosomes from mesangial cells that were treated with patient-derived gdIgA1 and IgA1 complexes. Proteomic analysis was performed to identify the exosome cargo and delineate the signaling pathways leading to maladaptive paracrine crosstalk. From our study, we aim to understand the molecular targets for therapeutic strategies in IgAN. Method Human mesangial cells were treated with/without IgA1, gdIgA1 or IgG for 24 h. Cell culture supernatant was collected for exosome isolations. Exosomes were analyzed by size, morphology and number using TEM and NanoFCM. Exosome protein extracts were processed for LC-Tandem mass spectrometry analysis using filter-aided sample preparation protocol. Proteomic analysis was performed using Qlucore omics explorer and pathway analysis using Ingenuity Pathway Analysis (IPA). Exosome marker expressions such as CD63, CD81 and FLOT1 and proteome validation was performed using Western blotting. Statistical analysis was performed using GraphPad Prism and values <0.05 were considered significant. Results We confirmed the release of exosomes by mesangial cells in response to IgA1/gdIgA1 complexes. Exosomes were fewer in unstimulated cells as compared to IgA1/gdIgA1 treated cells. They showed round morphology with a dense core and uniform size distribution. Exosomes from treated cells showed a mean size of about 75 nm and concentration in the range of 1-2 × 10^10 particles/ml. While untreated cells showed a mean size of about 100 nm and concentration of about 1 × 10^9 particles/ml. We confirmed the protein expression of exosome markers such as CD63, CD81 and FLOT1. For the first time, we demonstrated global proteomics on exosomes from mesangial cells followed by pathway analysis. We have identified 89 unique proteins and pathways in exosomes derived from gdIgA1 treated mesangial cells that could mediate pathological signaling in the glomerulus in comparison to exosomes derived from IgA1 treated mesangial cells. Conclusion Our study provides an understanding of the pathological crosstalk signaling through exosomes and key molecular targets for IgAN.

The role of exosomes in the diagnostics and treatment of immune mediated skin disoders, wounds and alopecia

Exosomes are microvesicles secreted by different cells that have the specificity and ability to transfer their cargo, including various regulatory molecules, to other cells. Exosomes cargo analysis considered to be a promising approach for diagnostics and therapeutic agents delivery into cells. Molecules derived from exosome cargo supposed to be plausible diagnostics criteria. This review provides up-to-date information on the exosomes origin and composition followed by a description of their diagnostic potential. New data summarized on the possibilities of exosomes application for the treatment of chronic immune-mediated cutaneous disorders, alopecia and cutaneous wound healing.

Fluorescent Chiral Quantum Dots to Unveil Origin-Dependent Exosome Uptake and Cargo Release.

Exosomes are promising nanocarriers for drug delivery. Yet, it is challenging to apply exosomes in clinical use due to the limited understanding of their physiological functions. While cellular uptake of exosomes is generally known through endocytosis and/or membrane fusion, the mechanisms of origin-dependent cellular uptake and subsequent cargo release of exosomes into recipient cells are still unclear. Herein, we investigated the intricate mechanisms of exosome entry into recipient cells and intracellular cargo release. In this study, we utilized chiral graphene quantum dots (GQDs) as representatives of exosomal cargo, taking advantage of the superior permeability of chiral GQDs into lipid membranes as well as their excellent optical properties for tracking analysis. We observed that the preferential cellular uptake of exosomes derived from the same cell-of-origin (intraspecies exosomes) is higher than that of exosomes derived from different cell-of-origin (cross-species exosomes). This uptake enhancement was attributed to receptor-ligand interaction-mediated endocytosis, as we identified the expression of specific ligands on exosomes that favorably interact with their parental cells and confirmed the higher lysosomal entrapment of intraspecies exosomes (intraspecies endocytic uptake). On the other hand, we found that the uptake of cross-species exosomes primarily occurred through membrane fusion, followed by direct cargo release into the cytosol (cross-species direct fusion uptake). We revealed the underlying mechanisms involved in the cellular uptake and subsequent cargo release of exosomes depending on their cell-of-origin and recipient cell types. Overall, this study envisions valuable insights into further advancements in effective drug delivery using exosomes, as well as a comprehensive understanding of cellular communication, including disease pathogenesis.

Targeting therapy for hepatocellular carcinoma by delivering microRNAs as exosomal cargo

Exosomes, the smallest extracellular vesicles, have gained significant attention as key mediators in intercellular communication, influencing both physiological and pathological processes, particularly in cancer progression. A recent review article by Wang et al was published in a timely manner to stimulate future research and facilitate practical developments for targeted treatment of hepatocellular carcinoma using exosomes, with a focus on the origin from which exosomes derive. If information about the mechanisms for delivering exosomes to specific cells is incorporated, the concept of targeted therapy for hepatocellular carcinoma using exosomes could be more comprehensively understood.

Abstract PO1-25-01: LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes

Abstract Background: Breast cancer (BC) is a leading cause of cancer death and despite numerous treatment advancements, metastatic BC is incurable. Extracellular vesicles (EVs) are lipid-delimited particles released from cells which contain bioactive protein and nucleic acid cargo. Numerous studies have shown their importance in facilitating communication between cancer cells and the tumour microenvironment, implicating them as mediators of many aspects of carcinogenesis. EV-mediated communication between BC and the innate immune system plays a crucial role in BC progression, metastasis and the maintenance of an immunosuppressive microenvironment. LMTK3 overexpression in BC promotes therapy resistance, disease progression and invasion and metastasis, leading to a poorer prognosis in patients. Analysis of deconvoluted TCGA data indicated that LMTK3 overexpression also correlates with exclusion of M1 macrophages from the tumour microenvironment. This study aimed to explore the role of LMTK3 in EV-mediated communication between BC and immune cells. Methods EVs were collected from conditioned media of wild-type and LMTK3-overexpressing BC cells through differential ultracentrifugation, then characterised by transmission electron microscopy, nanoparticle tracking analysis, quantitative mass spectrometry and Western blotting. Monocytes were treated with EVs and proteomic and phosphoproteomic changes were analysed. 3D coculture models of BC and immune cells were used to assess monocyte and macrophage infiltration, polarisation and anti-cancer activity. Immunofluorescence and flow cytometry was used to assess macrophage polarisation. Bioinformatic analysis of TCGA datasets validated the findings in a clinical setting and the data was also validated in vivo, using syngeneic immunocompetent mouse models. Results LMTK3 overexpression in BC cells promotes the upregulation of a subpopulation of large (80-150nm) CD81+ EVs which are significantly enriched in oncogenic and immunoregulatory proteins, including LDHB, ARHGEF15, PSAT1 and HIC2. EVs from LMTK3-overexpressing but not control BC cells strongly reduce the ability of monocytes to infiltrate BC tumour spheroids and migrate through Matrigel. Proteomics analysis of the monocytes showed an enrichment in M2 polarisation proteins and a downregulation of proteins essential for migration, including S100A8, KLF13 and TRPC3. LMTK3 overexpression in BC also promotes M2 polarisation and exclusion of M1 macrophages in vivo. Summary LMTK3 overexpression in BC correlates with poor prognosis and reduced overall survival. We hypothesise that this is, in part, due to monocyte M2 polarisation and exclusion of M1 macrophages from the TME, mediated by LMTK3-dependent alterations in EV cargo, contributing to tumour immune evasion. Citation Format: Mark Samuels, William Jones, Charlotte Turner, Georgios Giamas. LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-01.

Exosomes as a therapeutic tool to promote neurorestoration and cognitive function in neurological conditions: Achieve two ends with a single effort.

Exosomes possess a significant role in intercellular communications. In the nervous system, various neural cells release exosomes that not only own a role in intercellular communications but also eliminate the waste of cells, maintain the myelin sheath, facilitate neurogenesis, and specifically assist in normal cognitive function. In neurological conditions including Parkinson's disease (PD), Alzheimer's disease (AD), traumatic brain injury (TBI), and stroke, exosomal cargo like miRNAs take part in the sequela of conditions and serve as a diagnostic tool of neurological disorders, too. Exosomes are not only a diagnostic tool but also their inhibition or administration from various sources like mesenchymal stem cells and serum, which have shown a worthy potential to treat multiple neurological disorders. In addition to neurodegenerative manifestations, cognitive deficiencies are an integral part of neurological diseases, and applying exosomes in improving both aspects of these diseases has been promising. This review discusses the status of exosome therapy in improving neurorestorative and cognitive function following neurological disease.

Innovative Diagnosis and Therapeutic Modalities: Engineered Exosomes in Autoimmune Disease.

Autoimmune diseases refer to a group of conditions where the immune system produces an immune response against self-antigens, resulting in tissue damage. These diseases have profound impacts on the health of patients. In recent years, with the rapid development in the field of biomedicine, engineered exosomes have emerged as a noteworthy class of biogenic nanoparticles. By precisely manipulating the cargo and surface markers of exosomes, engineered exosomes have gained enhanced anti-inflammatory, immunomodulatory, and tissue reparative abilities, providing new prospects for the treatment of autoimmune diseases. Engineered exosomes not only facilitate the efficient delivery of bioactive molecules including nucleic acids, proteins, and cytokines, but also possess the capability to modulate immune cell functions, suppress inflammation, and restore immune homeostasis. This review mainly focuses on the applications of engineered exosomes in several typical autoimmune diseases. Additionally, this article comprehensively summarizes the current approaches for modification and engineering of exosomes and outlines their prospects in clinical applications. In conclusion, engineered exosomes, as an innovative therapeutic approach, hold promise for the management of autoimmune diseases. However, while significant progress has been made, further rigorous research is still needed to address the challenges that engineered exosomes may encounter in the therapeutic intervention process, in order to facilitate their successful translation into clinical practice and ultimately benefit a broader population of patients.

Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

Applications of Exosomal miRNAs from Mesenchymal Stem Cells as Skin Boosters.

The skin is the outer layer of the human body, and it is crucial in defending against injuries and damage. The regenerative capacity of aging and damaged skin caused by exposure to external stimuli is significantly impaired. Currently, the rise in average life expectancy and the modern population's aesthetic standards have sparked a desire for stem-cell-based therapies that can address skin health conditions. In recent years, mesenchymal stem cells (MSCs) as therapeutic agents have provided a promising and effective alternative for managing skin regeneration and rejuvenation, attributing to their healing capacities that can be applied to damaged and aged skin. However, it has been established that the therapeutic effects of MSC may be primarily mediated by paracrine mechanisms, particularly the release of exosomes (Exos). Exosomes are nanoscale extracellular vesicles (EVs) that have lipid bilayer and membrane structures and can be naturally released by different types of cells. They influence the physiological and pathological processes of recipient cells by transferring a variety of bioactive molecules, including lipids, proteins, and nucleic acids such as messenger RNAs (mRNAs) and microRNAs (miRNAs) between cells, thus playing an important role in intercellular communication and activating signaling pathways in target cells. Among them, miRNAs, a type of endogenous regulatory non-coding RNA, are often incorporated into exosomes as important signaling molecules regulating protein biosynthesis. Emerging evidence suggests that exosomal miRNAs from MSC play a key role in skin regeneration and rejuvenation by targeting multiple genes and regulating various biological processes, such as participating in inflammatory responses, cell migration, proliferation, and apoptosis. In this review, we summarize the recent studies and observations on how MSC-derived exosomal miRNAs contribute to the regeneration and rejuvenation of skin tissue, with particular attention to the applications of bioengineering methods for manipulating the miRNA content of exosome cargo to improve their therapeutic potential. This review can provide new clues for the diagnosis and treatment of skin damage and aging, as well as assist investigators in exploring innovative therapeutic strategies for treating a multitude of skin problems with the aim of delaying skin aging, promoting skin regeneration, and maintaining healthy skin.

The protein composition of exosomes released by prostate cancer cells is distinctly regulated by androgen receptor-antagonists and -agonist to stimulate growth of target cells.

Prostate cancer (PCa) is a prevalent malignancy in men worldwide, ranking as the second leading cause of cancer-related death in Western countries. Various PCa hormone therapies, such as androgen receptor (AR)-antagonists or supraphysiological androgen level (SAL) reduce cancer cell proliferation. However, treated cells may influence the growth of neighboring cells through secreted exosomes in the tumor microenvironment (TME). Here, the change of protein content of exosomes secreted from PCa cells through treatment with different AR-antagonists or SAL has been analyzed. Isolation of exosomes via ultracentrifugation of treated human PCa LNCaP cells with AR-agonist and various AR-antagonists; analysis of cellular senescence by detection of senescence associated beta galactosidase activity (SA β-Gal); Western blotting and immunofluorescence staining; Mass spectrometry (MS-spec) of exosomes and bioinformatic analyses to identify ligand-specific exosomal proteins. Growth assays to analyze influence of exosomes on non-treated cells. MS-spec analysis identified ligand-specific proteins in exosomes. One thousand seventy proteins were up- and 52 proteins downregulated by SAL whereas enzalutamide upregulated 151 proteins and downregulated 42 exosomal proteins. The bioinformatic prediction indicates an up-regulation of pro-proliferative pathways. AR ligands augment hub factors in exosomes that include AKT1, CALM1, PAK2 and CTNND1. Accordingly, functional assays confirmed that the isolated exosomes from AR-ligand treated cells promote growth of untreated PCa cells. The data suggest that the cargo of exosomes is controlled by AR-agonist and -antagonists and distinct among the AR-antagonists. Further, exosomes promote growth that might influence the TME. This finding sheds light into the complex interplay between AR signaling and exosome-mediated communication between PCa cells.

Albumen exosomes alleviate LPS-induced inflammation of intestinal epithelial cells via miR-22/ATM/p53/NF-κB axis

Biological macromolecules identified in albumen were found benefit to intestinal health, whether albumen contains exosomes and function of their cargos in intestinal inflammation remain unknown. This study aimed to investigate characteristics and cargos of albumen exosomes, as well as their potential roles in alleviating inflammation in intestinal epithelial cells. Our results demonstrated that albumen contains exosomes that are cup-shaped morphology vesicles with diameter ranging from 50 to 200 nm. There were 278 miRNAs and 45 proteins with higher expression levels in albumen exosomes, and they were mainly involved in immune responses and programmed cell death pathways, including apoptosis and p53 signaling pathway. LPS induced overexpression of pro-inflammatory cytokines IL-1β and TNF-α and excessive apoptosis, which could be reversed by albumen exosomes. The beneficial effects of exosomes could be mainly attributed to miRNA cargos and their inhibition on inflammatory response signaling pathways (p53 and NF-κB pathways). Mechanically, exosome miR-22 targeted ATM and inhibited p53/NF-κB pathway, alleviating LPS-induced overexpression of Caspase-3 and Bax, and inflammatory response. Collectively, albumen exosomes alleviate inflammation of intestinal epithelial cells via miR-22/ATM/p53/NF-κB axis and these findings may provide theoretical basis to the potential application of albumen exosomes for intestinal inflammation.

343 Development of osteoclast derived exosomes for vascular calcification therapy

OBJECTIVES/GOALS: The global incidence of calcific aortic valve disease (CAVD) increased 3.5-fold since 1990. No preventative or therapeutic pharmaceutical therapies exist for CAVD. We will establish the therapeutic potential of osteoclast-derived exosomes though characterization of contents and mechanisms of action to protect against mineralization. METHODS/STUDY POPULATION: Exosomes were purified from conditioned media collected from murine myeloid precursor cells, RAW264.7 (control), and osteoclasts induced to differentiate from RAW264.7 cells (OD). Protein content of exosomes was determined using proteomic analyses. Nucleic acid contents will be identified by sequencing mRNA, miRNA, and DNA. The calcification prevention and reabsorption abilities of control and OD exosomes will be tested using human valvular interstitial cells (VIC) and smooth muscle cell calcification assays and acellular osteologic disc assays, respectively. Comparison between cellular and acellular systems will help identify mechanisms of action, and demonstrate potential therapeutic viability of OD exosomes in preventative vs resorptive treatments. RESULTS/ANTICIPATED RESULTS: OD exosomes, but not control exosomes, prevented calcification in VIC in vitro. OD exosomes contained osteoclast-specific proteins including TRAP, MMP6, cathepsin K, and bone reabsorption factors including V type proton pumps, ATPases, and integrins. These genes are also involved in resorptive activities, and were highly upregulated in OD compared to control exosomes. We anticipate miRNA signatures associated with mineral resorption will also be present. Increased knowledge of exosome cargo will illuminate their mechanism of action and allow future work to engineer increased efficacy. We also anticipate a therapeutic response when OD exosomes are applied after calcification has begun, showing exosomes promote calcium reabsorption. DISCUSSION/SIGNIFICANCE: Establishing therapeutic potential and examining mechanisms of action will pave the way for OD exosomes as a CAVD treatment. Analysis of exosome contents will determine active molecules to be enhanced in future studies. This work will lay a foundation for moving into aortic valve organoid models, which are accepted by the FDA for preclinical trials.

Abstract 2990: Differential micro-RNA expression in circulating exosomes of pancreatic ductal adenocarcinoma patients

Abstract Pancreatic ductal Adenocarcinoma (PDAC) is a highly lethal solid tumor with 5-year survival rate of 5-8%. Extracellular vesicles like exosomes circulating in the blood are crucial mediators of intracellular communication, immunomodulation and tumor progression. The microRNAs being the major cargo of exosomes, are the primary carriers of intracellular information. The role of exosomal-miRNA (exoMIR’s) in regulating the pathogenesis of tumors is well-established in oncology but its potential is still unexplored. To the best of our knowledge no study has yet explored the contrasting exoMIR expression in cancer patients before and after their surgery. In this study, differential exoMIR’s profile of three pancreatic ductal adenocarcinoma patients at three stages: pre-surgery, post-surgery, and follow-up was established using small RNA sequencing on the Illumina platform. In blood samples taken before and after surgery total of 181, 201 and 201 microRNAs were dysregulated in circulating exosomes of three different patients viz. P_1, P_9 & P_11 (Patient identifiers) respectively. Out of the total dysregulated microRNAs in post-op samples 76 were upregulated and 105 downregulated in P_1, 95 upregulated and 106 downregulated in P_9 & 69 upregulated and 132 downregulated in P_11 as compared to their Pre surgery samples. Further, when exoMIR’s of post-op samples were compared to that of follow-up samples after 6 months of surgery their expression profile changed notably such that a total of 184, 157 & 190 microRNAs dysregulated in follow-up of P_1, P_9 and P_11 respectively. Out of the total dysregulated microRNAs in follow up samples 93 were upregulated and 91 downregulated in P_1, 69 upregulated and 88 downregulated in P_9 & 96 upregulated and 94 downregulated in P_11 as compared to their post-op samples. On Venn diagram analysis 27 microRNAs were found to be commonly dysregulated in all 3 patients before surgery and 22 common micro RNAs were again dysregulated even after 6 months of surgery. Bioinformatic analysis of these differentially expressed miRNAs and their respective protein targets revealed that their role in oncogenic molecular pathways is very prominent, thus highlighting their potential use in cancer therapeutics, diagnostics as well as prognosis. Citation Format: Rupinder Kaur, Sandeep Bhoriwal, Promod Kumar Gautam. Differential micro-RNA expression in circulating exosomes of pancreatic ductal adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2990.

Abstract 3015: HD-PTP mediates tumor suppression via modulation of anti-tumor immunity

Abstract The role of circulating extracellular vesicles in cancer biology is an area of intense research interest. Here, we reveal a tumor suppressor mechanism of HD-PTP (His Domain Protein Tyrosine Phosphatase, encoded by PTPN23), a protein associated with ESCRT (endosomal sorting complex required for transport), a complex that controls endosome sorting and exosome secretion. While loss of HD-PTP is known to be tumorigenic in mice and is frequently observed in human cancers including Triple Negative Breast Cancer (TNBC), the relevant pathways have remained obscure. We find that inhibition of HD-PTP in vivo promotes tumor progression selectively in a syngeneic, immune-competent TNBC model but not in a matched immune-deficient model. Through systematic profiling of the exosomal proteome in HD-PTP proficient and deficient cells, we identify PD-L1 as a key exosomal cargo selectively enriched due to loss of HD-PTP. Mechanistically, we show that inhibition of HD-PTP results in PD-L1 stabilization, its co-localization with the exosomal marker CD63, the enrichment of endosomal PD-L1 and subsequent increase in ExoPD-L1 release. Exosomes produced post HD-PTP loss inhibits antigen-dependent T cell activity in vitro, and HD-PTP inhibition in a syngeneic TNBC model promotes non-cell-autonomous, exosome- and PD-L1-dependent tumor progression, associated with reduced T cell infiltration and altered macrophage polarization. Consistent with these findings, low HD-PTP levels in human solid tumors are associated with decreased tumor infiltrating lymphocytes, poor response to immune checkpoint inhibition and inferior overall survival. Taken together, our studies unveil a novel tumor suppressor pathway controlling PD-L1 trafficking and ExoPD-L1 secretion, highlighting the critical roles of HD-PTP and ESCRT in anti-tumor immunity. Citation Format: Chenxu Guo, Sheng Sun, Nayana Thimmiah, Agustina Maccio, Siang Koh, Leif Ellisen. HD-PTP mediates tumor suppression via modulation of anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3015.

Decrease of exosomal miR-21-5p and the increase of CD62p+ exosomes are associated with the development of sepsis in polytraumatized patients

Sepsis as a severe systemic inflammation leads oftentimes to organ dysfunction and subsequently to death. In polytrauma patients, septic complications represent with 45% the predominant cause of late death and are responsible for extremely high costs in the healthcare system. Therefore, clinicians have to detect as early as possible the begin of sepsis to improve the patient's outcome. One new promising diagnostic tool to diagnose septic complications in polytraumatized patients are exosomes.Plasma samples from polytraumatized patients (Injury Severity Score (ISS) ≥16) which developed sepsis (n = 10) and without sepsis (n = 10), were collected at emergency room (ER), 24h and 5 days after trauma. The EVs subpopulations were investigated by a bead-based multiplex flow cytometry measurement of surface epitopes and were compared with plasma EVs from healthy controls (n = 10). Moreover, exosomal cytokine concentrations were measured via high-sensitive ELISA and were correlated with systemic concentrations. For miRNA cargo analysis, we analysed the miRNAs miR-1298-5p, miR-1262, miR-125b-5p, miR-92a-3p, miR-93-5p, miR-155-5p and miR-21-5p and compared their exosomal concentrations by means of RT-qPCR.CD62p + exosomes were significantly increased in septic polytrauma-patients (p ≤ 0.05), while CD40+exosomes, as well as CD49e + exosomes were diminished (p ≤ 0.05). Furthermore, we observed that the exosomal IL-6 concentration reflects the systemic IL-6 concentration (r2 = 0.63) and did not significantly alter between patients with and without sepsis. The exosomal IL-10 concentration seemed to be constant in all patients and healthy controls. We observed that a decrease of miR-21-5p in exosomes was associated with the development of sepsis (p ≤ 0.05), while exosomal miR-93-5p, miR-155-5p and miR-92a-3p were not specifically altered in septic patients.Taken together, the present study in polytraumatized patients demonstrated that the development of sepsis is associated with an increase of CD62p + exosomes. Furthermore, the exosomal cargo was changed in septic patients: miR-21-5p was diminished.

Extracellular vesicles as a new frontier of diagnostic biomarkers in osteosarcoma diseases: a bibliometric and visualized study.

The use of liquid biopsy in cancer research has grown exponentially, offering potential for early detection, treatment stratification, and monitoring residual disease and recurrence. Exosomes, released by cancer cells, contain tumor-derived materials and are stable in biofluids, making them valuable biomarkers for clinical evaluation. Bibliometric research on osteosarcoma (OS) and exosome-derived diagnostic biomarkers is scarce. Therefore, we aimed to conduct a bibliometric evaluation of studies on OS and exosome-derived biomarkers. Using the Web of Science Core Collection database, Microsoft Excel, the R "Bibliometrix" package, CiteSpace, and VOSviewer software, quantitative analyses of the country, author, annual publications, journals, institutions, and keywords of studies on exosome-derived biomarkers for OS from 1995 to 2023 were performed. High-quality records (average citation rate ≥ 10/year) were filtered. The corresponding authors were mainly from China, the USA, Australia, and Canada. The University of Kansas Medical Center, National Cancer Center, Japan, and University of Kansas were major institutions, with limited cooperation reported by the University of Kansas Medical Center. Keyword analysis revealed a shift from cancer progression to mesenchymal stem cells, exosome expression, biogenesis, and prognostic biomarkers. Qualitative analysis highlighted exosome cargo, including miRNAs, circRNAs, lncRNAs, and proteins, as potential diagnostic OS biomarkers. This research emphasizes the rapid enhancement of exosomes as a diagnostic frontier, offering guidance for the clinical application of exosome-based liquid biopsy in OS, contributing to the evolving landscape of cancer diagnosis.

Nerve Growth Factor-Preconditioned Mesenchymal Stem Cell-Derived Exosome-Functionalized 3D-Printed Hierarchical Porous Scaffolds with Neuro-Promotive Properties for Enhancing Innervated Bone Regeneration.

The essential role of the neural network in enhancing bone regeneration has often been overlooked in biomaterial design, leading to delayed or compromised bone healing. Engineered mesenchymal stem cells (MSCs)-derived exosomes are becoming increasingly recognized as potent cell-free agents for manipulating cellular behavior and improving therapeutic effectiveness. Herein, MSCs are stimulated with nerve growth factor (NGF) to regulate exosomal cargoes to improve neuro-promotive potential and facilitate innervated bone regeneration. In vitro cell experiments showed that the NGF-stimulated MSCs-derived exosomes (N-Exos) obviously improved the cellular function and neurotrophic effects of the neural cells, and consequently, the osteogenic potential of the osteo-reparative cells. Bioinformatic analysis by miRNA sequencing and pathway enrichment revealed that the beneficial effects of N-Exos may partly be ascribed to the NGF-elicited multicomponent exosomal miRNAs and the subsequent regulation and activation of the MAPK and PI3K-Akt signaling pathways. On this basis, N-Exos were delivered on the micropores of the 3D-printed hierarchical porous scaffold to accomplish the sustained release profile and extended bioavailability. In a rat model with a distal femoral defect, the N-Exos-functionalized hierarchical porous scaffold significantly induced neurovascular structure formation and innervated bone regeneration. This study provided a feasible strategy to modulate the functional cargoes of MSCs-derived exosomes to acquire desirable neuro-promotive and osteogenic potential. Furthermore, the developed N-Exos-functionalized hierarchical porous scaffold may represent a promising neurovascular-promotive bone reparative scaffold for clinical translation.

Human adenovirus type 3 restores pharmacologically inhibited exosomal cargo in lung carcinoma cells.

Introduction: Drug repurposing is fast growing and becoming an attractive approach for identifying novel targets, such as exosomes for cancer and antiviral therapy. Exosomes are a specialized class of extracellular vesicles that serve as functional mediators in intercellular communication and signaling that are important in normal physiological functions. A continuously growing body of evidence has established a correlation between the abnormal release of exosomes with various viral disease pathologies including cancer. Cells that are virus-infected release exosomes known to influence the process via the loading and transfer of viral components, such as miRNA, small (s) RNA, DNA, and proteins. Inhibition of exosome release may abate the spread and severity of viral infection, thus making exosomes an attractive target for antiviral therapies. We previously demonstrated the pharmacological inhibition of exosomes. Methods: Herein, we used a cell-based assay to determine the effect of Human adenovirus type 3 (HAdV3) on the exosome inhibition process by azole and Heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points. Results: HAdV3 activities led to increased total sRNA, DNA, and exosome particle concentrations via particle tracking in the presence of Climbazole and Heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALG-2 interacting protein X (ALIX), and tetraspanin (CD63), (p < 0.05) and upregulated transcription factor interferon regulatory factor (IRF) 8 in the presence of HAdV3 after 24hours (h) of treatment. Whereas higher concentrations of Climbazole and Heparin sodium salt were found to inhibit total exosome protein (p < 0.001) and exo-RNA (p < 0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome related DNA damage/repair signaling proteins. Blocking exosome secretion partially obstructed viral entry. Immunological studies revealed that HAdV3 fiber protein levels in A549 cells were reduced at all concentrations of Climbazole and Heparin and both multiplicities of infections (p < 0.001). Discussion: Our findings suggest that while HAdV may bolster inhibited exosome content and release when modulating certain activities of the endosomal pathway mediators, HAdV entry might be constrained by the activities of these pharmacological agents.