iPSC-derived lung and lung cancer organoid model to evaluate cisplatin encapsulated autologous iPSC-derived mesenchymal stromal cell-isolated extracellular vesicles.

BACKGROUND: Lung cancer remains a leading cause of cancer-related mortality globally. Although recent therapeutic advancements have provided targeted treatment approaches, the development of resistance and systemic toxicity remain primary concerns. Extracellular vesicles (EVs), especially those derived from mesenchymal stromal cells (MSC), have gained attention as promising drug delivery systems, offering biocompatibility and minimal immune responses. Recognizing the limitations of conventional 2D cell culture systems in mimicking the tumor microenvironment, this study aims to describe a proof-of-principle approach for using patient-specific organoid models for both lung cancer and normal lung tissue and the feasibility of employing autologous EVs derived from induced pluripotent stem cell (iPSC)-MSC in personalized medicine approaches. METHODS: First, we reprogrammed healthy fibroblasts into iPSC. Next, we differentiated patient-derived iPSC into branching lung organoids (BLO) and generated patient-matched lung cancer organoids (LCO) from patient-derived tumor tissue. We show a streamlined process of MSC differentiation from iPSC and EV isolation from iPSC-MSC, encapsulated with 0.07 µg/mL of cytotoxic agent cisplatin and applied to both organoid models. Cytotoxicity of cisplatin and cisplatin-loaded EVs was recorded with LDH and CCK8 tests. RESULTS: Fibroblast-derived iPSC showed a normal karyotype, pluripotency staining, and trilineage differentiation. iPSC-derived BLO showed expression of lung markers, like TMPRSS2 and MUC5A while patient-matched LCO showed expression of Napsin and CK5. Next, we compared the effects of iPSC-MSC derived EVs loaded with cisplatin against empty EVs and cisplatin alone in lung cancer organoid and healthy lung organoid models. As expected, we found a cytotoxic effect when LCO were treated with 20 µg/mL cisplatin. Treatment of LCO and BLO with empty EVs resulted in a cytotoxic effect after 24 h. However, EVs loaded with 0.07 µg/mL cisplatin failed to induce any cytotoxic effect in both organoid models. CONCLUSION: We report on a proof-of-principle pipeline towards using autologous or allogeneic iPSC-MSC EVs as drug delivery tests for lung cancer in future. However, due to the time and labor-intensive processes, we conclude that this pipeline might not be feasible for personalized approaches at the moment.

Extracellular vesicles reshape the tumor microenvironment to improve cancer immunotherapy: Current knowledge and future prospects.

Tumor immunotherapy has revolutionized cancer treatment, but limitations remain, including low response rates and immune complications. Extracellular vesicles (EVs) are emerging as a new class of therapeutic agents for various diseases. Recent research shows that changes in the amount and composition of EVs can reshape the tumor microenvironment (TME), potentially improving the effectiveness of immunotherapy. This exciting discovery has sparked clinical interest in using EVs to enhance the immune system's response to cancer. In this Review, we delve into the world of EVs, exploring their origins, how they're generated, and their complex interactions within the TME. We also discuss the crucial role EVs play in reshaping the TME during tumor development. Specifically, we examine how their cargo, including molecules like PD-1 and non-coding RNA, influences the behavior of key immune cells within the TME. Additionally, we explore the current applications of EVs in various cancer therapies, the latest advancements in engineering EVs for improved immunotherapy, and the challenges faced in translating this research into clinical practice. By gaining a deeper understanding of how EVs impact the TME, we can potentially uncover new therapeutic vulnerabilities and significantly enhance the effectiveness of existing cancer immunotherapies.

Tetrahydroisoquinoline reduces angiogenesis by interacting myeloma cells with HUVECs mediated by extracellular vesicles.

Multiple myeloma (MM) is a neoplastic condition resulting from the uncontrolled expansion of B-cell-derived plasma cells. The importance of angiogenesis in MM development has also been demonstrated. Extracellular vesicles (EVs) have vital functions in interactions between neighboring cells, such as angiogenesis. The objective of this in vitro study was to examine the transfection and angiogenesis effects of MM-EVs on endothelial cells (ECs) upon treatment with Tetrahydroisoquinoline (THIQ) as a bioactive organic compound derivative from isoquinoline. Following treatment of multiple myeloma cells (U266) with THIQ, MM-EVs were harvested and transmigrated to human umbilical vein endothelial cells (HUVEC) in a co-culture model. EVs transmigration was traced by flow cytometry. Correspondingly, the expression of angiogenic genes and/or proteins in U266 cells and HUVECs was measured by RT-PCR and ELISA methods. Likewise, the proliferation and migration of HUVECs treated with THIQ-treated MM-EVs were visualized and estimated by performing both tube formation and scratch wound healing methods. Surprisingly, the anti-angiogenic effect of THIQ-treated MM-EVs was evident by the decreased expression of CD34, VEGFR2, and IL-6 at the mRNA and/or protein levels after internalization of MM-EVs in HUVEC. Finally, tube formation and scratch wound healing experiments showed inhibition of HUVEC cell proliferation and migration by THIQ-treated MM-EVs compared to control MM-EVs. MM-EVs derived from THIQ-treated myeloma cells (U266) inhibited angiogenesis in HUVECs. This phenomenon is coordinated by the internalized THIQ-treated MM-EVs in HUVECs, and ultimately the reduction of angiogenic factors and inhibition of tube formation and scratch wound healing.

Extracellular vesicles isolated from curcumin-medium weakened RKO cell proliferation and migration.

Background: Curcumin (Cur) is a natural phytochemical that is expected to become an indispensable drug for the treatment of colorectal cancer. A comprehensive understanding of the anti-tumor mechanism of Cur will provide a better reference for its clinical application. This study aimed to examine the effects of extracellular vesicles (EVs) isolated from Cur-medium on RKO colorectal cancer cell proliferation, apoptosis, and migration. Methods: RKO cells were cultured in various concentrations of Cur-medium, and the EVs were isolated from the Cur-medium. The EVs were identified by transmission electron microscopy and western blotting. The effects of the EVs on RKO cell proliferation, apoptosis, and migration were analyzed, as was the expression of proliferating cell nuclear antigen (PCNA), Bax, vimentin, and E-cadherin. The expression of nuclear factor κB (NF-κB) p65 in the EVs was also detected. Results: Our results showed that the EVs isolated from the Cur-medium weakened RKO cell proliferation and migration but had no effect on cell apoptosis. Cur suppressed the expression of NF-κB p65 in the EVs. Overall, this study revealed that Cur exerts anti-tumor effects by suppressing NF-κB p65 in EVs to weaken RKO cell proliferation and migration. Conclusions: In conclusion, the packaging of Cur into EVs is expected to become an indispensable treatment of colorectal cancer in the future.

Establishing the capacity to monitor proteins relevant to the study of drug exposure and response using liver-derived extracellular vesicles.

AIMS: Drug exposure and response is determined by pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Interindividual differences in abundance of drug metabolizing enzymes (DMEs) and drug target proteins underpin PK and PD variability and impact treatment efficacy and tolerability. Extracellular vesicles (EVs) carry protein cargo inherited from originating cells and may be useful for defining differences in key proteins related to hepatic drug metabolism and the treatment of metabolic-associated fatty liver disease (MAFLD). We sought to quantify these proteins in liver-derived EVs and establish the profile relative to paired tissue. METHODS: EVs were recovered from human liver tissue samples (LT-EV, n = 11). Targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays were employed for absolute quantification of proteins in EV isolates and matched liver tissue. RESULTS: DMEs and MAFLD drug targets were readily detected and quantified in LT-EVs. Twelve of 15 DMEs exhibited moderate to strong correlation (Spearman ⍴ = 0.618-0.973) between tissue and EVs. Correlation in protein abundance was influenced by the extent of extra-hepatic expression of the target. CONCLUSIONS: This study provides evidence that key proteins related to PK and PD profiles can be measured in liver-derived EVs and abundance of liver-enriched DMEs are robustly correlated between paired tissue and EVs. The robust detection of protein markers related to drug PD profile in MAFLD opens the possibility to track within-subject changes in MAFLD and lays the foundation for future development of a liver-derived EV liquid biopsy to assess markers of drug exposure and response in vivo.

Circulating Liquid Biopsy Biomarkers in Glioblastoma: Advances and Challenges.

Gliomas, particularly glioblastoma (GBM), represent the most prevalent and aggressive tumors of the central nervous system (CNS). Despite recent treatment advancements, patient survival rates remain low. The diagnosis of GBM traditionally relies on neuroimaging methods such as magnetic resonance imaging (MRI) or computed tomography (CT) scans and postoperative confirmation via histopathological and molecular analysis. Imaging techniques struggle to differentiate between tumor progression and treatment-related changes, leading to potential misinterpretation and treatment delays. Similarly, tissue biopsies, while informative, are invasive and not suitable for monitoring ongoing treatments. These challenges have led to the emergence of liquid biopsy, particularly through blood samples, as a promising alternative for GBM diagnosis and monitoring. Presently, blood and cerebrospinal fluid (CSF) sampling offers a minimally invasive means of obtaining tumor-related information to guide therapy. The idea that blood or any biofluid tests can be used to screen many cancer types has huge potential. Tumors release various components into the bloodstream or other biofluids, including cell-free nucleic acids such as microRNAs (miRNAs), circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), proteins, extracellular vesicles (EVs) or exosomes, metabolites, and other factors. These factors have been shown to cross the blood-brain barrier (BBB), presenting an opportunity for the minimally invasive monitoring of GBM as well as for the real-time assessment of distinct genetic, epigenetic, transcriptomic, proteomic, and metabolomic changes associated with brain tumors. Despite their potential, the clinical utility of liquid biopsy-based circulating biomarkers is somewhat constrained by limitations such as the absence of standardized methodologies for blood or CSF collection, analyte extraction, analysis methods, and small cohort sizes. Additionally, tissue biopsies offer more precise insights into tumor morphology and the microenvironment. Therefore, the objective of a liquid biopsy should be to complement and enhance the diagnostic accuracy and monitoring of GBM patients by providing additional information alongside traditional tissue biopsies. Moreover, utilizing a combination of diverse biomarker types may enhance clinical effectiveness compared to solely relying on one biomarker category, potentially improving diagnostic sensitivity and specificity and addressing some of the existing limitations associated with liquid biomarkers for GBM. This review presents an overview of the latest research on circulating biomarkers found in GBM blood or CSF samples, discusses their potential as diagnostic, predictive, and prognostic indicators, and discusses associated challenges and future perspectives.

Are Treatment Patterns for Endophthalmitis After Cataract Surgery Following the EVS Trial Recommendations? An IRIS® Registry Analysis.

OBJECTIVE: Evaluate whether treatment patterns for endophthalmitis after cataract surgery in American Academy of Ophthalmology IRIS® Registry (Intelligent Research in Sight) patients are in line with evidence-based guidelines established by the 1995 Endophthalmitis Vitrectomy Treatment Study (EVS), which showed that patients who present with light perception vision (LP) have better visual outcomes with immediate vitrectomy (VIT) compared with vitreous tap with antibiotic injection (TAP). DESIGN: Retrospective cohort study SUBJECTS: IRIS Registry patients undergoing cataract surgery between 2014 and 2022 (identified by CPT codes), presenting with endophthalmitis (identified by ICD 10 codes) within 42 days post-cataract surgery, and having a record of being treated with VIT or TAP on the same or one day following endophthalmitis diagnosis were identified. METHODS: Potential covariates of age, sex, race, ethnicity, geographic region, insurance status, and visual acuity on the day of endophthalmitis diagnosis were evaluated using multivariable logistic regression. MAIN OUTCOME MEASURE: Treatment with VIT or TAP RESULTS: Of the 2425 patients who met the inclusion criteria, 14% (345) underwent VIT and 86% (2080) underwent TAP. 80% of patients (1946) presented with endophthalmitis within 14 days from cataract surgery (median = 6 days). Notably, 66% (173/263) of the patients presenting with LP vision underwent TAP instead of VIT. In a multivariable logistic regression model, receiving VIT instead of TAP was positively associated with poor vision at endophthalmitis presentation (LP - OR, 5.3 [CI: 2.9-10.5]; Counting Fingers, Hand Motion - OR 1.8 [CI: 1.0-3.6]) vs. [20/20-20/40] vision; Asian vs. White race (OR, 2.6 [CI: 1.2-5.1]); Hispanic vs. Non-Hispanic ethnicity (OR, 1.9 [CI, 1.0-3.2]); living in the West (OR, 1.5 [CI, 1.1-2.2]) and Midwest (OR- 1.4 [CI, 1.0-2.0]) (vs. South), but not with age, sex, and insurance coverage (P>0.05). CONCLUSIONS: In the IRIS Registry, treatment patterns for post-cataract surgery endophthalmitis did not match evidence-based recommendations of the EVS, a randomized controlled clinical trial. More work is needed to evaluate whether the current treatment patterns are optimal for patients with post-cataract surgery endophthalmitis.

Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs' dose-response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs' biotherapeutics.

Engineered small extracellular vesicle-mediated ferroptosis: A new frontier in cancer immunotherapy.

Ferroptosis is a novel iron-dependent form of cell death discovered in recent years, characterized by the accumulation of ferrous iron, the production of reactive oxygen species (ROS) through the Fenton reaction, and lipid peroxidation, ultimately leading to the disruption of the antioxidant system and cell membrane damage. Extensive research has found that ferroptosis plays a significant role in regulating tumor cell immune evasion, tumor development, and remodeling the tumor microenvironment. Small Extracellular vesicles (sEVs), carrying various bioactive molecules (ncRNA, DNA, proteins), are key nanoscale mediators of intercellular communication. Increasing evidence confirms that EVs can regulate the ferroptosis pathway in tumors, promoting tumor cell immune evasion and reshaping the tumor microenvironment. This article aims to comprehensively review the key mechanisms by which sEVs mediate ferroptosis in cancer and provide new insights into targeting tumor immunotherapy.

Unveiling the Cutting-Edge Impact of Polarized Macrophage-Derived Extracellular Vesicles and MiRNA Signatures on TGF-ß Regulation within Lung Fibroblasts.

Depending on local cues, macrophages can polarize into classically activated (M1) or alternatively activated (M2) phenotypes. This study investigates the impact of polarized macrophage-derived Extracellular Vesicles (EVs) (M1 and M2) and their cargo of miRNA-19a-3p and miRNA-425-5p on TGF-ß production in lung fibroblasts. EVs were isolated from supernatants of M0, M1, and M2 macrophages and quantified using nanoscale flow cytometry prior to fibroblast stimulation. The concentration of TGF-ß in fibroblast supernatants was measured using ELISA assays. The expression levels of miRNA-19a-3p and miRNA-425-5p were assessed via TaqMan-qPCR. TGF-ß production after stimulation with M0-derived EVs and with M1-derived EVs increased significantly compared to untreated fibroblasts. miRNA-425-5p, but not miRNA-19a-3p, was significantly upregulated in M2-derived EVs compared to M0- and M1-derived EVs. This study demonstrates that EVs derived from both M0 and M1 polarized macrophages induce the production of TGF-ß in fibroblasts, with potential regulation by miRNA-425-5p.

Metformin modifies plasma microbial-derived extracellular vesicles in polycystic ovary syndrome with insulin resistance.

INTRODUCTION: This study investigated changes in plasma microbial-derived extracellular vesicles (EVs) in patients with polycystic ovary syndrome and insulin resistance (PCOS-IR) before and after metformin treatment, and aimed to identify bacterial taxa within EVs that were biologically and statistically significant for diagnosis and treatment. METHODS: The case-control study was conducted at Xiamen Chang Gung Hospital, Hua Qiao University. Plasma samples were collected from five PCOS-IR patients of childbearing age before and after 3 months of metformin treatment, and the samples were sequenced. The diversity and taxonomic composition of different microbial communities were analyzed through full-length 16 S glycosomal RNA gene sequencing. RESULTS: After metformin treatment, fasting plasma glucose levels and IR degree of PCOS-IR patients were significantly improved. The 16 S analysis of plasma EVs from metformin-treated patients showed higher microbial diversity. There were significant differences in EVs derived from some environmental bacteria before and after metformin treatment. Notably, Streptococcus salivarius was more abundant in the metformin-treated group, suggesting it may be a potential probiotic. DISCUSSION: The study demonstrated changes in the microbial composition of plasma EVs before and after metformin treatment. The findings may offer new insights into the pathogenesis of PCOS-IR and provide new avenues for research.

Chronic Lymphocytic Leukemia (CLL)-Derived Extracellular Vesicles Educate Endothelial Cells to Become IL-6-Producing, CLL-Supportive Cells.

We hypothesized that via extracellular vesicles (EVs), chronic lymphocytic leukemia (CLL) cells turn endothelial cells into CLL-supportive cells. To test this, we treated vein-derived (HUVECs) and artery-derived (HAOECs) endothelial cells with EVs isolated from the peripheral blood of 45 treatment-naïve patients. Endothelial cells took up CLL-EVs in a dose- and time-dependent manner. To test whether CLL-EVs turn endothelial cells into IL-6-producing cells, we exposed them to CLL-EVs and found a 50% increase in IL-6 levels. Subsequently, we filtered out the endothelial cells and added CLL cells to this IL-6-enriched medium. After 15 min, STAT3 became phosphorylated, and there was a 40% decrease in apoptosis rate, indicating that IL-6 activated the STAT3-dependent anti-apoptotic pathway. Phospho-proteomics analysis of CLL-EV-exposed endothelial cells revealed 23 phospho-proteins that were upregulated, and network analysis unraveled the central role of phospho-ß-catenin. We transfected HUVECs with a ß-catenin-containing plasmid and found by ELISA a 30% increase in the levels of IL-6 in the culture medium. By chromatin immunoprecipitation assay, we observed an increased binding of three transcription factors to the IL-6 promoter. Importantly, patients with CLL possess significantly higher levels of peripheral blood IL-6 compared to normal individuals, suggesting that the inducers of endothelial IL-6 are the neoplastic EVs derived from the CLL cells versus those of healthy people. Taken together, we found that CLL cells communicate with endothelial cells through EVs that they release. Once they are taken up by endothelial cells, they turn them into IL-6-producing cells.

Advancements in Personalized CAR-T Therapy: Comprehensive Overview of Biomarkers and Therapeutic Targets in Hematological Malignancies.

Chimeric antigen receptor T-cell (CAR-T) therapy is a novel anticancer therapy using autologous or allogeneic T-cells. To date, six CAR-T therapies for specific B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and multiple myeloma (MM) have been approved by the Food and Drug Administration (FDA). Significant barriers to the effectiveness of CAR-T therapy include cytokine release syndrome (CRS), neurotoxicity in the case of Allogeneic Stem Cell Transplantation (Allo-SCT) graft-versus-host-disease (GVHD), antigen escape, modest antitumor activity, restricted trafficking, limited persistence, the immunosuppressive microenvironment, and senescence and exhaustion of CAR-Ts. Furthermore, cancer drug resistance remains a major problem in clinical practice. CAR-T therapy, in combination with checkpoint blockades and bispecific T-cell engagers (BiTEs) or other drugs, appears to be an appealing anticancer strategy. Many of these agents have shown impressive results, combining efficacy with tolerability. Biomarkers like extracellular vesicles (EVs), cell-free DNA (cfDNA), circulating tumor (ctDNA) and miRNAs may play an important role in toxicity, relapse assessment, and efficacy prediction, and can be implicated in clinical applications of CAR-T therapy and in establishing safe and efficacious personalized medicine. However, further research is required to fully comprehend the particular side effects of immunomodulation, to ascertain the best order and combination of this medication with conventional chemotherapy and targeted therapies, and to find reliable predictive biomarkers.

Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma.

Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.

Chemo-small extracellular vesicles released in cisplatin-resistance ovarian cancer cells are regulated by the lysosomal function.

Chemoresistance is a common problem in ovarian cancer (OvCa) treatment, where resistant cells, in response to chemotherapy, secrete small extracellular vesicles (sEVs), known as chemo-sEVs, that transfer resistance to recipient cells. sEVs are formed as intraluminal vesicles (ILVs) within multivesicular endosomes (MVEs), whose trafficking is regulated by Ras-associated binding (RAB) GTPases that mediate sEVs secretion or lysosomal degradation. A decrease in lysosomal function can promote sEVs secretion, but the relationship between MVEs trafficking pathways and sEVs secretion in OvCa chemoresistance is unclear. Here, we show that A2780cis cisplatin (CCDP) resistant OvCa cells had an increased number of MVEs and ILVs structures, higher levels of Endosomal Sorting Complex Required for Transport (ESCRTs) machinery components, and RAB27A compared to A2780 CDDP-sensitive OvCa cells. CDDP promoted the secretion of chemo-sEVs in A2780cis cells, enriched in DNA damage response proteins. A2780cis cells exhibited poor lysosomal function with reduced levels of RAB7, essential in MVEs-Lysosomal trafficking. The silencing of RAB27A in A2780cis cells prevents the Chemo-EVs secretion, reduces its chemoresistance and restores lysosomal function and levels of RAB7, switching them into an A2780-like cellular phenotype. Enhancing lysosomal function with rapamycin reduced chemo-sEVs secretion. Our results suggest that adjusting the balance between secretory MVEs and lysosomal MVEs trafficking could be a promising strategy for overcoming CDDP chemoresistance in OvCa.

The Role of the Intestinal Flora and Its Derivatives in Neurocognitive Disorders: A Narrative Review from Surgical Perspective.

Perioperative neurocognitive dysfunction is a significant concern for population health, impacting postoperative recovery and increasing the financial burden on patients. With an increasing number of surgical procedures being performed, the prevention and management of perioperative neurocognitive dysfunction have garnered significant attention. While factors such as age, lifestyle, genetics, and education are known to influence the development of cognitive dysfunction, recent research has highlighted the role of the gut microbiota in neurological health. An increased abundance of pro-inflammatory gut microbiota can trigger and worsen neuroinflammation, neuronal cell damage, and impaired cellular autophagy. Moreover, the inflammation-promoting gut microbiota can disrupt immune function, impair neuroautophagy, and affect the production and circulation of extracellular vesicles and neurotransmitters. These factors collectively play a role in the onset and advancement of cognitive impairment. This narrative review delves into the molecular mechanisms through which gut microbiota and their derivatives contribute to cognitive impairment, focusing on the impact of anesthesia surgery, changes in gut microbial populations, and perioperative cognitive impairment associations. The study suggests that alterations in the abundance of various bacterial species and their metabolites pre- and post-surgery may be linked to postoperative cognitive impairment. Furthermore, the potential of probiotics or prebiotics in addressing cognitive impairment is discussed, offering a promising avenue for investigating the treatment of perioperative neurocognitive disorders.

Microglia Morphological Response to Mesenchymal Stromal Cell Extracellular Vesicles Demonstrates EV Therapeutic Potential for Modulating Neuroinflammation.

Background: Mesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation-relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs. Results: Using an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-γ/IFN-α stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism. Conclusion: This study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphological approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.

Pushing Forward the DNA Walkers in Connection with Tumor-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are microparticles released from cells in both physiological and pathological conditions and could be used to monitor the progression of various pathological states, including neoplastic diseases. In various EVs, tumor-derived extracellular vesicles (TEVs) are secreted by different tumor cells and are abundant in many molecular components, such as proteins, nucleic acids, lipids, and carbohydrates. TEVs play a crucial role in forming and advancing various cancer processes. Therefore, TEVs are regarded as promising biomarkers for the early detection of cancer in liquid biopsy. However, the currently developed TEV detection methods still face several key scientific problems that need to be solved, such as low sensitivity, poor specificity, and poor accuracy. To overcome these limitations, DNA walkers have emerged as one of the most popular nanodevices that exhibit better signal amplification capability and enable highly sensitive and specific detection of the analytes. Due to their unique properties of high directionality, flexibility, and efficiency, DNA walkers hold great potential for detecting TEVs. This paper provides an introduction to EVs and DNA walker, additionally, it summarizes recent advances in DNA walker-based detection of TEVs (2018-2024). The review highlights the close relationship between TEVs and DNA walkers, aims to offer valuable insights into TEV detection and to inspire the development of reliable, efficient, simple, and innovative methods for detecting TEVs based on DNA walker in the future.

Adult cardiomyocytes-derived EVs for the treatment of cardiac fibrosis.

Cardiac fibrosis is a common pathological feature of cardiovascular diseases that arises from the hyperactivation of fibroblasts and excessive extracellular matrix (ECM) deposition, leading to impaired cardiac function and potentially heart failure or arrhythmia. Extracellular vesicles (EVs) released by cardiomyocytes (CMs) regulate various physiological functions essential for myocardial homeostasis, which are disrupted in cardiac disease. Therefore, healthy CM-derived EVs represent a promising cell-free therapy for the treatment of cardiac fibrosis. To this end, we optimized the culture conditions of human adult CMs to obtain a large yield of EVs without compromising cellular integrity by using a defined combination of small molecules. EVs were isolated by ultracentrifugation, and their characteristics were analysed. Finally, their effect on fibrosis was tested. Treatment of TGFß-activated human cardiac fibroblasts with EVs derived from CMs using our culture system resulted in a decrease in fibroblast activation markers and ECM accumulation. The rescued phenotype was associated with specific EV cargo, including multiple myocyte-specific and antifibrotic microRNAs, although their effect individually was not as effective as the EV treatment. Notably, pathway analysis showed that EV treatment reverted the transcription of activated fibroblasts and decreased several signalling pathways, including MAPK, mTOR, JAK/STAT, TGFß, and PI3K/Akt, all of which are involved in fibrosis development. Intracardiac injection of CM-derived EVs in an animal model of cardiac fibrosis reduced fibrotic area and increased angiogenesis, which correlated with improved cardiac function. These findings suggest that EVs derived from human adult CMs may offer a targeted and effective treatment for cardiac fibrosis, owing to their antifibrotic properties and the specificity of cargo.

Biological Activities of Citrus-Derived Extracellular Vesicles on Human Cells: The Role of Preservation.

Extracellular vesicles (EVs) have been identified as important mediators for cell-to-cell communication. Citrus-based EVs in particular offer an excellent platform for nutraceutical delivery systems, as their endemic cargo includes micronutrients (e.g., ascorbic acid), which contribute to their antioxidant capacity. Despite being extensively investigated as to their therapeutic and diagnostic potential, their cargo is inherently unstable and thus directly affected by their storage and preservation. In this study, EVs were isolated from citrus fruit using tangential flow filtration and evaluated for their physicochemical characteristics, antioxidant activity and effects on human cells. To assess how their isolation and preservation methods affect these properties, the EVs were tested immediately after isolation (from fresh and freeze-thawed juices) or following freeze-drying. A measurable biological effect of cryoprotection on citrus-derived EVs was evident, whether during or after isolation. This was more pronounced in the cell-based assays, ranging from -4% to +32% in human skin fibroblast proliferation. Nevertheless, the effects on human cancer cells varied depending on the cell line. Although these results should be considered preliminary observations, subject to further investigation, it is safe to state that any type of preservation is expected to impact the EVs' biological activity.