Exosomal microRNAs Research Articles

Dumbbell Dual-Hairpin Triggered DNA Nanonet Assembly for Cascade-Amplified Sensing of Exosomal MicroRNA.

Exosomal microRNAs (miRNAs) are valuable biomarkers closely associated with cancer progression. Therefore, sensitive and specific exosomal miRNA biosensing has been employed for cancer diagnosis, prognosis, and prediction. In this study, a miRNA-based DNA nanonet assembly strategy is proposed, enabling the biosensing of exosomal miRNAs through dumbbell dual-hairpin under isothermal enzyme-free conditions. This strategy dexterously designs a specific dumbbell dual-hairpin that can selectively recognize exosomal miRNA, inducing conformational changes to cascade-generated X-shaped DNA structures, facilitating the extension of the X-shaped DNA in three-dimensional space, ultimately forming a DNA nanonet assembly. On the basis of the target miRNA, our design enriches the fluorescence signal through the cascade assembly of DNA nanonet and realizes the secondary signal amplification. Using exosomal miR-141 as the target, the resultant fluorescence sensing demonstrates an impressive detection limit of 57.6 pM and could identify miRNA sequences with single-base variants with high specificity. Through the analysis of plasma and urine samples, this method effectively distinguishes between benign prostatic hyperplasia, prostate cancer, and metastatic prostate cancer. Serving as a novel noninvasive and accurate screening and diagnostic tool for prostate cancer, this dumbbell dual-hairpin triggered DNA nanonet assembly strategy is promising for clinical applications.

Human Breast Milk Exosomal miRNAs are Influenced by Premature Delivery and Affect Neurodevelopment.

This study investigates the exosomal microRNA (miRNA) profiles of term and preterm breast milk, including the most abundant and differentially expressed (DE) miRNAs, and their impact on neurodevelopment in infants. Mature milk is collected from the mothers of term and preterm infants. Using high-throughput sequencing and subsequent data analysis, exosomal miRNA profiles of term and preterm human breast milk (HBM) are acquired and it is found that the let-7 and miR-148 families are the most abundant miRNAs. Additionally, 23 upregulated and 15 downregulated miRNAs are identified. MiR-3168 is the most upregulated miRNA in preterm HBM exosome, exhibiting targeting activity toward multiple genes involved in the SMAD and MAPK signaling pathways and playing a crucial role in early neurodevelopment. Additionally, the effects of miR-3168 on neurodevelopment is confirmed and it is determined that it is an essential factor in the differentiation of neural stem cells (NSCs). This study demonstrates that miRNA expression in breast milk exosomes can be influenced by preterm delivery, thereby potentially impacting neurodevelopment in preterm infants.

Goat milk exosomal microRNAs alleviate LPS-induced intestinal inflammation in mice

Intestinal inflammation is a common digestive system disease. Milk-derived exosomes can participate in intercellular communication and transport a variety of bioactive components, and the microRNAs (miRNAs) they carry play important roles in a variety of biological processes in the body. At present, the preventive effect and mechanism of action of goat milk exosomes and their derived miRNAs on intestinal inflammation are still unclear. In this study, the protective effect of goat milk exosomes on LPS-induced intestinal inflammation was investigated using mouse intestinal inflammation model and IEC-6 cell inflammation model. Small RNA sequencing was used to analyze the miRNA expression profile of goat milk exosomes. In this study, C-Exo and M-Exo alleviated intestinal inflammation by reducing the LPS-induced release of proinflammatory cytokines, inhibiting the increase in the NLRP3 protein and the activation of the TLR4/NFκB signaling pathway. C-Exo has a more significant inhibitory effect on them, and better therapeutic efficacy than M-Exo. Notably, the target genes of miRNAs in C-Exo and M-Exo were significantly enriched in immune-related pathways. Furthermore, their derived miR-26a-5p and miR-30a-5p were found to ameliorate the IEC-6 inflammatory response. These findings suggest that miRNAs in goat milk exosomes have the potential to attenuate LPS-induced intestinal inflammation.

Transferrin-conjugated magnetic nanoparticles for the isolation of brain-derived blood exosomal MicroRNAs: A novel approach for Parkinson's disease diagnosis

BackgroundBrain-derived exosomes circulate in the bloodstream and other bodily fluids, serving as potential indicators of neurological disease progression. These exosomes present a promising avenue for the early and precise diagnosis of neurodegenerative conditions. Notably, miRNAs found in plasma extracellular vesicles (EVs) offer distinct diagnostic benefits due to their stability, abundance, and resistance to breakdown. ResultsIn this study, we introduce a method using transferrin conjugated magnetic nanoparticles (TMNs) to isolate these exosomes from the plasma of patients with neurological disorders. This TMNs technique is both quick (<35 min) and cost-effective, requiring no high-priced ingredients or elaborate equipment for EV extraction. Our method successfully isolated EVs from 33 human plasma samples, including those from patients with Parkinson's disease (PD), Multiple Sclerosis (MS), and Dementia. Using quantitative polymerase chain reaction (PCR) analysis, we evaluated the potential of 8 exosomal miRNA profiles as biomarker candidates. Six exosomal miRNA biomarkers (miR-195-5p, miR-495-3p, miR-23b-3P, miR-30c-2-3p, miR-323a-3p, and miR-27a-3p) were consistently linked with all stages of PD. SignificanceThe TMNs method provides a practical, cost-efficient way to isolate EVs from biological samples, paving the way for non-invasive neurological diagnoses. Furthermore, the identified miRNA biomarkers in these exosomes may emerge as innovative tools for precise diagnosis in neurological disorders including PD.

Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Abstract LB013: Characterizing the role of exosomal miRNAs in metastasis

Abstract Background: Exosomal microRNAs (Ex-miRs) play a pivotal role in intercellular communication, being transported via extracellular vesicles. In cancer, Ex-miRs influence tumor progression by regulating key cellular processes such as proliferation, angiogenesis, and metastasis. Their role in mediating communication between cancer cells and the tumor microenvironment highlights their significance as potential diagnostic and therapeutic targets. Methodology: In this study, we aimed to characterize the role of Ex-miRs in influencing the pre-metastatic niche (PMN) across 7 tumor types. We extracted high-confidence Ex-miRs (Log FC &amp;gt;= 2 in exosomes relative to expectation) and their targets (experimentally proven and targeted by at least 2 Ex-miRs) from various studies. Subsequently, we conducted gene ontology analysis to identify enriched pathways and selected the top 100 high-confidence Ex-miR targets based on the frequency of their appearance in the enriched pathways. These top 100 targets were consistently used throughout the analysis. Results: Pathway enriched among the top Ex-miR targets included common ones such as “wound healing” and “regulation of epithelial cell proliferation”, as well as cancer-specific, like “regulation of angiogenesis in kidney” (KIRC), “ossification” in lung (LUAD), and “positive regulation of cytokine production” in pancreatic cancer (PAAD). Similarly, 'Pathways in cancer' and 'MicroRNAs in cancer' ranked among the top 10 enriched KEGG pathways for all cancer types. Ex-miR targets exhibited cancer-specific downregulation of gene expression in TCGA-Normal Adjacent Tumors (NATs), taken as proxy for PMN, compared to healthy tissue samples in GTEx. We further examined the 3’UTR GC content of these targets and found that Ex-miR targets are GC-rich, consistent with the previous findings stating that Ex-miR bind to the 3’UTR region of the target gene, downregulating their expression. Fisher’s exact test revealed a significant overlap of cancer-specific tumor suppressor genes (TSG) with Ex-miR targets. Cox regression indicated that Ex-miR targets are associated with better survival in certain cancer types, such as BRCA, COAD and KIRC. Lastly, a Support Vector Machine (SVM) model using Ex-miR target gene expression classified responders and non-responders to neoadjuvant chemotherapy with a high AUROC compared to other gene signatures. Conclusion: Our study unveils the pivotal role of exosomal microRNAs in shaping the PMN in diverse cancers, underscoring the diagnostic and therapeutic potential of Ex-miRs. Citation Format: Piyush Agrawal, Gulden Olgun, Arashdeep Singh, Vishaka Gopalan, Sridhar Hannenhalli. Characterizing the role of exosomal miRNAs in metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB013.

Study on exosomes for identifying bipolar disorder in early stage: A cross-sectional and validation study protocol.

The difficulty is remained to accurately distinguish bipolar disorder (BD) from major depressive disorder (MDD) in early stage, with a delayed diagnosis for 5-10 years. BD patients are often treated with antidepressants systematically due to being diagnosed with MDD, affecting the disease course and clinical outcomes. The current study aims to explore the role of plasma exosomes as biomarker to distinguish BD from MDD in early stage. Two stages are included. The first stage is a cross-sectional study, comparing the concentrations of plasma exosome microRNA and related proteins among BD group, MDD group, and healthy controls (HC) group (n=40 respectively), to identify target biomarkers preliminarily. The "Latent Class Analysis" and "Receiver Operating Characteristic" analysis will be performed to determine the optimal concentration range for each biomarker. The second stage is to validate target markers in subjects, coming from an ongoing study focusing on patients with a first depressive episode. All target biomarkers will be test in plasma samples reserved at the initial stage to detect whether the diagnosis indicated by biomarker level is consistent with the diagnosis by DSM-5. Furthermore, the correlation between specific biomarkers and the manic episode, suicidal ideation, and adverse reactions will also be observed. Exosome-derived microRNA and related proteins have potential in serving as a good medium for exploring mental disorders because it can pass through the blood-brain barrier bidirectionally and convey a large amount of information stably. Improving the early diagnosis of BD would help implement appropriate intervention strategy as early as possible and significantly reduce the burden of disease.

496 Urinary Exosomal MicroRNA as Early Markers of Diabetic Kidney Disease in African American Adults

OBJECTIVES/GOALS: This study aimed to characterize urinary exosomal miRNA content in African American adults with diabetic kidney disease. METHODS/STUDY POPULATION: Male and female participants between the ages of 18 and 65 were recruited from the Diabetes Treatment Center and the Nephrology Clinic at the Howard University Hospital. Exosomes were isolated from cleared urine of healthy controls (n=3), type 2 diabetics (n=3), and participants with chronic kidney disease (n=3). The purity and size of isolated microparticles was evaluated using NanoSight technology (30nm to 120nm size range) and western blot analysis for exosome-specific markers (TSG101 and CD81) RESULTS/ANTICIPATED RESULTS: Expression of 5 selected microRNAs, miR-4534, miR-320c, miR-451, miR-362-3p and miR-877-3p were evaluated by qRT-PCR. miR-4534 and miR-451 was increased between healthy controls and the type diabetic group. MiR-320c was increased in the CKD group, in comparison to healthy controls. Conversely, there was no difference in miR-877-5p between the three groups. DISCUSSION/SIGNIFICANCE: These findings will provide insight into the use of circulating miRNAs as early markers of DKD, ultimately creating more effective treatments and preventive measures.

Nascent shifts in renal cellular metabolism, structure, and function due to chronic empagliflozin in prediabetic mice.

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

Low levels of microRNA-21 in neutrophil-derived exosomes may contribute to airway smooth muscle hyperproliferation in horses with severe asthma.

Neutrophilic inflammation is associated with the degree of airway obstruction in severe equine asthma (SEA), but the contribution of these leukocytes to bronchial remodeling remains ill defined. Neutrophils could cause structural alterations of the airways by the release of exosomes, a type of cell-derived nanoparticles that can modify the biology of local and distant cells. Neutrophil-derived exosomes have been shown to increase airway smooth muscle (ASM) cell proliferation in humans and horses. Therefore, this study aimed to identify neutrophil exosomal microRNAs (miRs) implicated in the regulation of ASM biology in SEA. 6 horses with SEA and 6 healthy controls. The expression of selected miRs in exosomes from peripheral neutrophils was studied by quantitative PCR. The effects of miR-21 transfection in ASM cells were evaluated by gene expression analysis and proliferation studies. The miR-21 was downregulated in neutrophil exosomes from SEA horses, and it attenuated the proliferation of ASM cells stimulated with lipopolysaccharide. The lower level of miR-21 in neutrophil-derived exosomes could contribute to ASM hyperproliferation, which could, in turn, promote the thickening of the bronchial wall in SEA.

Exosomal miRNA Changes Associated with Restoration to Sinus Rhythm in Atrial Fibrillation Patients.

We aimed to identify serum exosomal microRNAs (miRNAs) associated with the transition from atrial fibrillation (AF) to sinus rhythm (SR) and investigate their potential as biomarkers for the early recurrence of AF within three months post-treatment. We collected blood samples from eight AF patients at Chang Gung Memorial Hospital in Taiwan both immediately before and within 14 days following rhythm control treatment. Exosomes were isolated from these samples, and small RNA sequencing was performed. Using DESeq2 analysis, we identified nine miRNAs (16-2-3p, 22-3p, 23a-3p, 23b-3p, 125a-5p, 328-3p, 423-5p, 504-5p, and 582-3p) associated with restoration to SR. Further analysis using the DIABLO model revealed a correlation between the decreased expression of miR-125a-5p and miR-328-3p and the early recurrence of AF. Furthermore, early recurrence is associated with a longer duration of AF, presumably indicating a more extensive state of underlying cardiac remodeling. In addition, the reads were mapped to mRNA sequences, leading to the identification of 14 mRNAs (AC005041.1, ARHGEF12, AMT, ANO8, BCL11A, DIO3OS, EIF4ENIF1, G2E3-AS1, HERC3, LARS, NT5E, PITX1, SLC16A12, and ZBTB21) associated with restoration to SR. Monitoring these serum exosomal miRNA and mRNA expression patterns may be beneficial for optimizing treatment outcomes in AF patients.

Exosomal miR-1470 is a diagnostic biomarker and promotes cell proliferation and metastasis in colorectal cancer.

In recent years,the lack of specific markers for the diagnosis of colorectal cancer has led to an upward trend in both morbidity and mortality from this condition. There is an urgent need to identify molecular biomarkers that contribute to early cancer detection. This study aimed to identify specific exosomal microRNAs that hold potential as diagnostic biomarkers for CRC. We screened for differentially expressed miRNAs using the CRC exosome dataset GSE39833. To validate the results in the public database, we collected serum from 168 CRC patients and 168 healthy volunteers. The expression levels of exosomal miR-1470 in healthy volunteers and CRC patients were analyzed using qRT-PCR. To evaluate the diagnostic potential of the selected miR-1470 in distinguishing CRC patients from healthy controls, we analyzed its receiver operating characteristic curve. To explore the biological functions of miR-1470 in CRC cell lines, we detected the miR-1470's ability to regulate the growth and metastasis of CRC cells by CCK8, transwell and other assays after transfection of miR-1470 in SW480, HCT-116 cells. Exosomal miR-1470 exhibited significant up-regulation in CRC patients compared to healthy volunteers. The ROC curve analysis revealed an area under the curve (AUC) of 0.74 (95% confidence interval: 0.6876-0.7920) for exosomal miR-1470, indicating its potential as a diagnostic biomarker. Furthermore, the expression level of miR-1470 in CRC patients showed correlations with age, metastasis, and HDL content. We overexpressed miR-1470 in CRC cell lines. CCK8 proliferation assay showed that miR-1470 promoted the proliferation ability of SW480 and HCT-116 cells. Transwell assay showed that miR-1470 promoted the migration and invasion ability of SW480 and HCT-116 cells. This suggested that non-invasive diagnosis of CRC is possible by detecting the level of miR-1470 in exosomes, which has important implications for early detection and treatment of this disease.

Exosomal microRNAs in Parkinson's disease: insights into biomarker potential and disease pathology.

Parkinson's disease (PD) is a prevalent neurodegenerative condition primarily affecting the elderly population. Despite its high incidence in aged individuals, there are no reliable blood-based biomarkers for clinical diagnosis of PD and early screening of susceptible individuals. Recent studies have revealed the significance of exosomes in mediating cell-to-cell communications by transferring bioactive molecules, such as proteins, nucleic acids (including miRNAs), lipids, and metabolites, between cells. Due to their ability to carry diverse molecular cargo and their involvement in various physiological and pathological processes, exosomes have gained significant attention as potential disease biomarkers. Notably, exosomes have the ability to cross the blood-brain barrier, and as a result, they can be found in circulating body fluids, including cerebrospinal fluid (CSF), serum, and plasma. Therefore, the identification of PD-specific exosomes in blood samples could be a promising avenue with biomarker potential for advancing clinical diagnosis and planning therapeutic strategies. This review highlights the current understanding of exosomal miRNAs in PD pathology, emphasising their potential for clinical utility as biomarkers even though several challenges may have to be overcome to precisely utilize exosomal miRNAs as biomarkers specific to PD.

Effects of microgravity on neural crest stem cells.

Exposure to microgravity (μg) results in a range of systemic changes in the organism, but may also have beneficial cellular effects. In a previous study we detected increased proliferation capacity and upregulation of genes related to proliferation and survival in boundary cap neural crest stem cells (BC) after MASER14 sounding rocket flight compared to ground-based controls. However, whether these changes were due to μg or hypergravity was not clarified. In the current MASER15 experiment BCs were exposed simultaneously to μg and 1 g conditions provided by an onboard centrifuge. BCs exposed to μg displayed a markedly increased proliferation capacity compared to 1 g on board controls, and genetic analysis of BCs harvested 5 h after flight revealed an upregulation, specifically in μg-exposed BCs, of Zfp462 transcription factor, a key regulator of cell pluripotency and neuronal fate. This was associated with alterations in exosome microRNA content between μg and 1 g exposed MASER15 specimens. Since the specimens from MASER14 were obtained for analysis with 1 week's delay, we examined whether gene expression and exosome content were different compared to the current MASER15 experiments, in which specimens were harvested 5 h after flight. The overall pattern of gene expression was different and Zfp462 expression was down-regulated in MASER14 BC μg compared to directly harvested specimens (MASER15). MicroRNA exosome content was markedly altered in medium harvested with delay compared to directly collected samples. In conclusion, our analysis indicates that even short exposure to μg alters gene expression, leading to increased BC capacity for proliferation and survival, lasting for a long time after μg exposure. With delayed harvest of specimens, a situation which may occur due to special post-flight circumstances, the exosome microRNA content is modified compared to fast specimen harvest, and the direct effects from μg exposure may be partially attenuated, whereas other effects can last for a long time after return to ground conditions.

Exosomal encapsulation of miR-3198 promotes proliferation and migration of trophoblasts in preeclampsia

PurposePreeclampsia (PE) is a vascular remodeling disorder cloesly linked to trophoblast dysfunction, involving defects in their proliferation, migration, and apoptosis. Maternal exosomal microRNAs (miRNAs) have been reported to play pivotal roles in the development of PE. However, the mechanism underlying the role of maternal exosomes in trophoblast dysfunction regarding the development of PE is poorly understood.MethodsPlasma exosomes from maternal peripheral blood were collected from pregnant women with PE and from those with normal pregnancy. Bioinformatics analysis was used to identify significantly differentially expressed miRNAs under these two conditions. The expression of the miR-3198 gene in plasma exosomes was detected using quantitative real-time polymerase chain reaction. Dual luciferase reporter assay was used to confirm binding of miR-3198 and 3′UTR region of WNT3. Cell proliferation was examined using the Cell Count Kit-8 and EdU assays, and flow cytometry was performed to detect apoptosis and cell cycle. Changes in cell migration were examined using transwell and scratch assays.ResultsPatients with PE showed decreased expression of plasma-derived exosomal miR-3198. The proliferation and migration abilities of HTR-8/SVneo and primary human trophoblast cells were both improved when cocultured with miR-3198-rich exosomes. Exposure to miR-3198-enriched exosomes facilitated cell cycle progression but reduced apoptosis in HTR-8/SVneo cells. Notably, overexpression of miR-3198 partially prevented the inhibitory effects of WNT3 on proliferation and migration in HTR-8/SVneo cells.ConclusionExosomal miR-3198 in the maternal peripheral blood may regulate the biological functions of trophoblasts by targeting WNT3 and influence the development of diseases of placental origin.Graphical

Abstract 6410: An exosomal microRNA signature predicts the efficacy of ramucirumab plus paclitaxel in advanced gastric cancer: Findings from the prospective IVY trial

Abstract Purpose: With rapid advances in the development of effective therapeutic regimens in patients with advanced gastric cancer, it is imperative to establish individualized treatment approaches that can help identify patient subsets that have the highest likelihood of response to a specific regimen. Although ramucirumab plus paclitaxel therapy is the standard second-line treatment for patients with advanced gastric cancer, biomarkers that can help predict therapeutic response are still lacking. This longitudinal study aimed to establish a liquid-biopsy assay that can predict response to ramucirumab plus paclitaxel therapy in patients with advanced gastric cancer. Experimental Design: A prospective observational multicenter study (named the IVY study) enrolled 115 patients who received ramucirumab plus paclitaxel treatment. We collected serum specimens prior to the initiation of the treatment regimen. Patients were divided into responders and non-responders according to the response evaluation criteria in solid tumors (RECIST). We performed small RNA-sequencing (sRNA-Seq) in a subset of 28 patients with and without response to identify exosomal microRNA-based biomarkers that allowed risk stratification and response prediction in patients with advanced gastric cancer. Results: The sRNA-Seq analysis and rigorous bioinformatic approaches using machine-learning algorithms allowed us to identify a panel of 10 exosomal microRNAs that robustly predicted resistance to ramucirumab plus paclitaxel treatment (AUC: 0.83). The panel of exosomal miRNAs was also shown to have a robust prognostic value for predicting survival outcomes in gastric cancer patients treated with ramucirumab plus paclitaxel, and Cox proportional hazards regression analysis showed that patients classified as high risk on the panel of exosomal miRNAs independent poor prognostic factor (OS: HR = 2.74, 95% CI = 1.56-4.81, P &amp;lt; 0.01, RFS: HR = 1.84, 95% CI = 1.16-2.92, P &amp;lt; 0.01). Following logistic regression and backward elimination, we trained a clinically feasible 5 exosomal miRNA signature by combining it with the Body Mass Index data, an independent clinical predictor (AUC: 0.84). We subsequently successfully validated the performance of this signature in the prospective clinical trial cohort (AUC: 0.87). Finally, we established a prognostic risk nomogram based on the circulating exosomal miRNA signature for a non-invasive and personalized pre-therapeutic selection approach. Conclusions: We have developed a novel liquid biopsy-based assay that allows robust prediction of response to ramucirumab plus paclitaxel treatment in gastric cancer patients, and our assay provides a framework for a personalized medicine approach that can provide risk-based management and treatment for each patient prior to the initiation of therapy. Citation Format: Katsutoshi Shoda, Caiming Xu, Yoh Asahii, Takeshi Nagasaka, Daisuke Ichikawa, Ajay Goel. An exosomal microRNA signature predicts the efficacy of ramucirumab plus paclitaxel in advanced gastric cancer: Findings from the prospective IVY trial [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 6410.

Abstract 5875: Upregulation of the RNA binding protein HuR by cancer-secreted exosome to mediate multidrug resistance

Abstract Background and Aim: Multidrug resistance (MDR) is an unresolved obstacle to cancer chemotherapy. It is usually caused by overexpression of drug efflux transporters including ABCG2. Exosomes are extracellular lipid vesicles released from all cells upon the fusion of multivesicular bodies with plasma membrane. Tumor-derived exosomes have been shown to mediate cell-to-cell transfer of oncogenic materials to promote drug resistance. Human antigen R (HuR) is an extensively studied RNA binding protein that regulates the stability, translation, and nucleus-to-cytoplasm shuttling of mRNAs. Numerous HuR-targeting mRNAs play crucial roles in tumorigenesis, invasion and metastasis. We have previously reported the induction of ABCG2 and MDR phenotype by HuR overexpression in colon cancer. HuR overexpression is linked with high-grade malignancy and poor prognosis in cancer patients. This study investigated the enrichment of HuR inducing microRNAs (miRNAs) in exosomes secreted from MDR colon cancer and its intracellular transfer to drive MDR. Method: Cancer exosome-mediated transfer of miRNAs from ABCG2-overexpressing resistant colon cancer cells S1M1-80 to a few sensitive colon cell lines was evaluated. Exosomes were isolated from S1M1-80 conditioned medium by differential centrifugation method. The uptake of fluorescence-labeled exosome from donor MDR cancer cells by recipient sensitive cells was visualized by fluorescence microscopy. MiRNA microarray was conducted to identify differentially expressed miRNAs in drug sensitive cells after incubation with S1M1-80-secreted exosomes. Western blot and qPCR analysis were conducted to measure HuR and ABCG2 expression. Result: Exosomes were isolated from resistant S1M1-80 cells, whose purity was confirmed by the presence of exosomal markers (Tsg101 and Alix) but absence of cellular marker (GRP78) by Western blot analysis. These exosomes reduced the apoptotic effect of SN-38 or 5-fluorouracil in sensitive colon cancer cell lines (HCT116, S1, and SW480) by inducing both HuR and ABCG2. A common upregulated miRNA (miR-155-5p) was identified by miRNA microarray in the sensitive cell lines after incubation with S1M1-80-derived exosomes. MiR-155-5p was found to induce HuR expression by promoting its translation. MiR-155-5p inhibitor was shown to reverse the upregulation of HuR in HCT116 cells after exosome incubation, which subsequently suppressed ABCG2 expression and the MDR phenotype. Furthermore, inhibition of exosome uptake by dynasore was found to prevent the induction of HuR/ABCG2, and re-sensitize the exosome-treated sensitive cells to SN38. Conclusion: MDR cells-derived exosomes may spread the drug resistance phenotype by transferring a miR-155-5p/HuR/ABCG2 regulatory machinery to sensitive cells. Inhibition of this exosomal transfer of regulatory materials may prevent the spread of MDR and sensitize cancer cells to chemotherapy. Citation Format: Kenneth K.W. To. Upregulation of the RNA binding protein HuR by cancer-secreted exosome to mediate multidrug resistance [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 5875.

Abstract 6389: An exosome-based liquid biopsy powered by machine learning predicts progression-free and overall survival before commencing first-line EGFR inhibitors for metastatic colorectal cancer: The EXONERATE Study

Abstract Introduction: We developed a liquid biopsy assay based on exosomal and cell-free microRNA (exo- and cf-miRNA, respectively) for patients with chemotherapy-naïve, metastatic, unresectable, RAS and BRAF wild-type colorectal cancer (mCRC), to predict progression-free (PFS) and overall survival (OS) before the commencement of first-line treatment with EGFR inhibitors (EXONERATE). Methods: Patients with mCRC were consecutively enrolled in two open-label, nationwide clinical trials and received first-line cetuximab or panitumumab. Cf- and exo-miRNA expression was quantified using RT-qPCR in plasma samples obtained before exposure to EGFR inhibitors. The prespecified primary endpoint was 12-month PFS, hierarchically tested in left-sided mCRC, right-sided mCRC, and the entire cohort. As secondary endpoints, the EXONERATE status was correlated with PFS and OS hazard ratios (HR). Results: After excluding those with RAS/BRAF mutations and unavailable samples, the complete analysis dataset comprised 120 patients (94 with left-sided and 26 with right-sided mCRC; 87 received cetuximab and 33 panitumumab). As expected, right-sided mCRC patients showed inferior PFS and OS outcomes (HR 1.80 [CI95% = 1.13 - 2.80] and 1.80 [CI95% = 1.00 - 3.27], respectively). Genome-wide small RNA sequencing on 20 patient samples identified 12 cf- and 14 exo-miRNA candidates, then narrowed to 8 and 9 based on best performance. We employed machine learning algorithms to develop two liquid biopsy assays, one based on cf- and the other on exo-miRNAs, predicting 12mPFS with AUC values of 0.84 and 0.81, respectively. These were then combined to establish the EXONERATE assay for left-sided mCRC (AUC = 0.89), successfully validated in right-sided mCRC patients (AUC= 0.84). Among left-sided mCRC patients, 35% were EXONERATE-high, exhibiting shorter median PFS (mPFS = 9.5 vs. 18.5 months, p&amp;lt;.001) and a higher mortality rate (HR 0.21 [CI95% = 0.10 - 0.42]). In the right-sided mCRC cohort, the EXONERATE-high panel robustly identified patients with shorter mPFS (8.6 vs. 23.7 months, p=.0042) and predicted 12mPFS with high accuracy (sensitivity 100%, NPV 81%, PPV 100%). Although in univariate analysis both the EXONERATE panel and sidedness showed associations with PFS and OS, in multivariate analysis only the EXONERATE panel could fully account for the differences in PFS and OS (HR 0.22 and HR 0.19, respectively). Hence, we employed the EXONERATE panel agnostically of tumor sidedness and observed that EXONERATE-high patients demonstrated poorer survival outcomes in mPFS (8.9 vs. 19.6 months, HR 0.27 [CI95% = 0.17 - 0.41]) and mOS (22.6 vs. &amp;gt;60 months, HR 0.18 [CI95% = 0.09 - 0.37]). Conclusion: The EXONERATE panel robustly predicted PFS and OS outcomes in both right- and left-sided mCRC patients before receiving either EGFR inhibitor. Citation Format: Xu Caiming, Alessandro Mannucci, Roy Souvick, Esposito Francis, Oliveres Helena, Alonso-Orduña Vicente, Escudero Pilar, Fernández-Martos Carlos, Salud Antonieta, Gallego Javier, Rodriguez Jose Ramon, Martín-Richard Marta M. Marta, Fernández-Plana Julen, Manzano Hermini, Aparicio Jorge, Feliu Jaime, Maurel Joan, Ajay Goel. An exosome-based liquid biopsy powered by machine learning predicts progression-free and overall survival before commencing first-line EGFR inhibitors for metastatic colorectal cancer: The EXONERATE Study [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 6389.

Exploring the role of exosomal MicroRNAs as potential biomarkers in preeclampsia.

The complex pathogenesis of preeclampsia (PE), a significant contributor to maternal and neonatal mortality globally, is poorly understood despite substantial research. This review explores the involvement of exosomal microRNAs (exomiRs) in PE, focusing on their impact on the protein kinase B (AKT)/hypoxia-inducible factor 1-α (HIF1α)/vascular endothelial growth factor (VEGF) signaling pathway as well as endothelial cell proliferation and migration. Specifically, this article amalgamates existing evidence to reveal the pivotal role of exomiRs in regulating mesenchymal stem cell and trophoblast function, placental angiogenesis, the renin-angiotensin system, and nitric oxide production, which may contribute to PE etiology. This review emphasizes the limited knowledge regarding the role of exomiRs in PE while underscoring the potential of exomiRs as non-invasive biomarkers for PE diagnosis, prediction, and treatment. Further, it provides valuable insights into the mechanisms of PE, highlighting exomiRs as key players with clinical implications, warranting further exploration to enhance the current understanding and the development of novel therapeutic interventions.

Dynamics of serum exosome microRNA profile altered by chemically induced estropause and rescued by estrogen therapy in female mice.

The decline in the ovarian reserve leads to menopause and reduced serum estrogens. MicroRNAs are small non-coding RNAs, which can regulate gene expression and be secreted by cells and trafficked in serum via exosomes. Serum miRNAs regulate tissue function and disease development. Therefore, the aim of this study was to identify miRNA profiles in serum exosomes of mice induced to estropause and treated with 17β-estradiol (E2). Female mice were divided into three groups including control (CTL), injected with 4-Vinylcyclohexene diepoxide (VCD), and injected with VCD plus E2 (VCD + E2). Estropause was confirmed by acyclicity and a significant reduction in the number of ovarian follicles (p < 0.05). Body mass gain during estropause was higher in VCD and VCD + E2 compared to CTL females (p = 0.02). Sequencing of miRNAs was performed from exosomes extracted from serum, and 402 miRNAs were detected. Eight miRNAs were differentially regulated between CTL and VCD groups, seven miRNAs regulated between CTL and VCD + E2 groups, and ten miRNAs regulated between VCD and VCD + E2 groups. Only miR-200a-3p and miR-200b-3p were up-regulated in both serum exosomes and ovarian tissue in both VCD groups, suggesting that these exosomal miRNAs could be associated with ovarian activity. In the hepatic tissue, only miR-370-3p (p = 0.02) was up-regulated in the VCD + E2 group, as observed in serum. Our results suggest that VCD-induced estropause and E2 replacement have an impact on the profile of serum exosomal miRNAs. The miR-200 family was increased in serum exosomes and ovarian tissue and may be a candidate biomarker of ovarian function.