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Highlights: SERPINE1 mediates the transfer of cancer-derived exosomal let-7 g-5p to promote macrophage M2 polarization. Exosomal let-7 g-5p drives M2 polarization by downregulating SOCS7 and relieving its inhibition of STAT3 phosphorylation. SERPINE1 promotes the transcription of let-7 g-5p by activating the JAK2/STAT3 signaling pathway.Background: Tumor-associated macrophages (TAMs), particularly M2-polarized TAMs, are significant contributors to tumor progression, immune evasion, and therapy resistance in gastric cancer (GC). Despite efforts to target TAM recruitment or depletion, clinical efficacy remains limited. Consequently, the identification of targets that specifically inhibit or reprogram M2-polarized TAMs presents a promising therapeutic strategy. Objective: This study aims to identify a dual-function target in GC cells that drives both malignant phenotypes and M2 macrophage polarization, revealing its molecular mechanisms to provide novel therapeutic targets for selectivly targeting M2-polarized TAMs in GC. Methods: Transcriptomic and clinical data from GC and adjacent tissues were utilized to identify mRNAs associated with high M2 macrophage infiltration and poor prognosis. Single-cell sequencing elucidated cell types expressing the target gene. Transwell co-culture and exosome intervention experiments demonstrated its role in M2 polarization. Small RNA sequencing of exosomes, western blotting, and CoIP assays revealed the molecular mechanisms underlying exosome-mediated M2 polarization. Protein array, ChIP and dual-luciferase reporter assays clarified the molecular mechanisms by which the target gene regulated exosomal miRNA. In vivo validation was performed using xenograft tumor models. Results: SERPINE1 was identified as a highly expressed mRNA in GC tissues and cells, significantly associated with advanced clinical stages, worse prognosis, and higher M2 macrophage infiltration in patients with GC. SERPINE1 overexpression in GC cells promoted tumor growth and M2 macrophage polarization. SERPINE1 facilitated the transfer of let-7 g-5p to macrophages via cancer-derived exosomes, inducing M2 polarization. Exosomal let-7 g-5p internalized by macrophages downregulated SOCS7 protein levels, disrupting its interaction with STAT3 and relieving the inhibition of STAT3 phosphorylation, thereby leading to STAT3 hyperactivation, which consequently drove M2 polarization. Additionally, in GC cells, elevated SERPINE1 expression activated JAK2, enhancing STAT3 binding to the let-7 g-5p promoter and promoting its transcription, thereby increasing let-7 g-5p levels in exosomes. Conclusion: GC cell-derived SERPINE1, functioning as a primary driver of GC growth and TAM M2 polarization, promotes M2 polarization through the regulation of exosomal let-7 g-5p transfer via autocrine activation of the JAK2/STAT3 signaling pathway. These findings elucidate a novel mechanism of SERPINE1-induced M2 polarization and highlight SERPINE1 as a promising target for advancing immunotherapy and targeted treatments in GC. [ABSTRACT FROM AUTHOR]

• Exosomal transfer of GC cell-derived miR-92b-5p facilitates TAMs M2 polarization through sustained STAT3 activation. • PLXNC1 upregulates exosomal miR-92b-5p expression derived from GC cells through activating MEK1/MSK1/CREB1 signaling pathway. • STAT3 achieves self-activation by mediating the transfer of GC cell-derived exosomal miR-92b-5p to macrophages. • EGCG derived from HDW targets STAT3 to inhibit M2 polarization induced by PLXNC1 -mediated GC cell-derived exosomes. M2-polarized tumor-associated macrophages (TAMs) predominate in tumor microenvironment (TME) and serve primary functions in tumor progression, including growth, angiogenesis, metastasis, immunosuppression, chemoresistance, and poor prognosis. The reversal of M2 polarization provides a new treatment strategy for cancer. Presently, the molecular mechanisms of M2 polarization have yet to be fully characterized, and there is a lack of effective therapeutic targets and drugs. Cancer cells initiate an immunosuppressive TME by recruiting macrophages and promoting M2 polarization through the secretion of inflammatory factors. Accordingly, blocking cancer cell-induced TAM M2 polarization may present a more effective strategy from the perspective of cancer cells. Hedyotis diffusa Willd (HDW) possesses immunomodulatory and antitumor properties, and is a precious and direct source of small molecule natural products with a dual function of inhibition of tumor growth and tumor cell-mediated M2 polarization. To identify a new target promoting gastric cancer (GC) cell growth and GC cell-mediated M2 polarization from mRNA profiles of GC cells treated with HDW injection (HDI) and to excavate a natural product from HDI that can regulate related mRNA and inhibit the aforementioned effects. RNA sequencing (RNA-seq) was used to analyze HDI-regulated differentially expressed mRNAs (HRmRNAs) in MKN45 cells. Weighted gene co-expression network analysis (WGCNA), univariate and multivariate Cox regression analysis, KM survival curves, and association analysis between HRmRNA and clinical characteristics/tumor infiltrating immune cells (TIICs) individually were utilized to screen out the target HRmRNA associated with prognosis and M2 macrophage infiltration in GC. shRNA lentiviral vectors were used for stably silencing, and transient overexpressing plasmids were constructed for overexpression. CCK8, EdU, colony formation, migration and invasion assays were used to validate the function of drugs and molecules in GC. HDI constituent analysis was performed using UHPLC-QE-MS. A network of HDI constituent-hub transcription factor (TF)-HRmRNA was constructed based on RNA-Seq, network pharmacology and TFs prediction. Exosome isolation and identification were performed using ultracentrifugation, NTA, TEM and western blot. Apoptosis and macrophage phenotypes were determined by flow cytometric analysis. Small RNA-Seq made exosomal miRNA identification. Small molecule interaction with targets were analyzed using molecular docking, SPR and CETSA. The direct relationship between transcription factors and promoters was verified using ChIP-QPCR and dual-luciferase reporter gene assay. A nude mice xenograft tumor model was established for vivo validation. HDI inhibited MKN45 cell proliferation, migration, invasion and promoted apoptosis. RNA-Seq identified 2583 HRmRNAs. PLXNC1 was screened out as the target HRmRNA associated with prognosis and M2 macrophage infiltration in GC. PLXNC1 promoted GC cell proliferation and facilitated TAMs M2 polarization by transferring GC cell-derived exosomal miR-92b-5p, inhibiting SOCS7-STAT3 interactions and subsequently activating STAT3 in macrophages. M2 TAMs induced by PLXNC1 -mediated GC cell-derived exosomes promoted GC cell migration and invasion. PLXNC1 regulated exosomal miR-92b-5p through the MEK1/MSK1/CREB1 pathway. STAT3 could transcriptionally regulate PLXNC1 expression in GC cells. The network of HDI constituent-hub TF-HRmRNA showed epigallocatechin gallate (EGCG) from HDI targeted STAT3 to transcriptionally regulate PLXNC1 expression. EGCG as a natural product directly bound to STAT3 to diminish its nuclear localization, resulting in the transcriptional repression of PLXNC1 and the reversal of M2 polarization induced by PLXNC1 -mediated GC cell-derived exosomes. PLXNC1 is a novel target exerting dual effects on GC cell proliferation and GC cell-mediated M2 polarization. EGCG derived from HDI inhibits GC cell proliferation and targets STAT3 to inhibit M2 polarization induced by PLXNC1 -mediated exosomes derived from GC cells, which may be a multi-target therapeutic agent for GC cell proliferation and immune microenvironment. [ABSTRACT FROM AUTHOR]

Multi-functionalized carbon nanomaterials have attracted interest owing to their excellent synergic properties, such as plasmon resonance energy transfer and surface-enhanced Raman scattering. Particularly, nanoparticle (NP)-decorated graphene (GRP) has been applied in various fields. In this study, silver NP (AgNP)- and magnetic iron oxide NP (IONP)-decorated GRP were prepared and utilized as biosensing platforms. In this case, AgNPs and GRP exhibit plasmonic properties, whereas IONPs exhibit magnetic properties; therefore, this hybrid nanomaterial could function as a magnetoplasmonic substrate for the magnetofluoro-immunosensing (MFI) system. Conversely, exosomes were recently considered high-potential biomarkers for the diagnosis of diseases. However, exosome diagnostic use requires complex isolation and purification methods. Nevertheless, we successfully detected a prostate-cancer-cell-derived exosome (PC-exosome) from non-purified exosomes in a culture media sample using Ag/IO-GRP and dye-tetraspanin antibodies (Ab). First, the anti-prostate-specific antigen was immobilized on the Ag/IO-GRP and it could isolate the PC-exosome from the sample via an external magnetic force. Dye-tetraspanin Ab was added to the sample to induce the sandwich structure. Based on the number of exosomes, the fluorescence intensity from the dye varied and the system exhibited highly sensitive and selective performance. Consequently, these hybrid materials exhibited excellent potential for biosensing platforms. [ABSTRACT FROM AUTHOR]

[Display omitted] • A high-performance g-C 3 N 4 /Ag hybrid SERS substrate was developed through a fast and easily controllable two-step electro-synthesis strategy. • The g-C 3 N 4 films possessed a highly ordered structure, providing sufficient sites for the even growth of Ag NPs through "Ag-N" bonds. • Accurate label-free detection of three early-stage cancers was achieved by combining SERS technology with an artificial intelligence (PCA-DL) model. Early surveillance and differentiation of cancer possesses remarkable clinical merit, but no effective protocol exists to reliably recognize multiple categories of early-stage cancers. In the present research, we investigate the viability in label-free inspection of cancers by combining surface-enhanced Raman spectroscopy (SERS) with principal component analysis (PCA)-deep learning (DL) to analyze exosomes. Based on a simple two-step electro-synthetical strategy, g-C 3 N 4 /Ag hybrid substrate with chemical enhancement ability and homogeneous electromagnetic hotspots was developed to directly collect the SERS spectra of various cancer cell-original exosomes (prostate, colon, and bladder) with high intensity and perfect resolution. Furthermore, a PCA-DL algorithm was constructed to delicately analyze the SERS spectra with similar characteristic peaks. Particularly, these cancer-derived exosomes were successfully identified with an accuracy of over 97.5 % and a specificity of over 97.6 % after capturing several key spectral features. In conclusion, the present work not only develops a promising SERS sensor for cancer surveillance, but also exploits a novel method to evaluate the spectral characteristics of exosomes. [ABSTRACT FROM AUTHOR]

The use of cancer-derived exosomes has been studied in several cancer types, but the cancer-targeting efficacy of glioma-derived exosomes has not been investigated in depth for malignant glioblastoma (GBM) cells. In this study, exosomes were derived from U87MG human glioblastoma cells, and selumetinib, a new anticancer drug, was loaded into the exosomes. We observed the tropism of GBM-derived exosomes in vitro and in vivo. We found that the tropism of GBM-derived exosomes is in contrast to the behavior of non-exosome-enveloped drugs and non-GBM-specific exosomes in vitro and in vivo in an animal GBM model. We found that the tropism exhibited by GBM-derived exosomes can be utilized to shuttle selumetinib, with no specific targeting moiety, to GBM tumor sites. Therefore, our findings indicated that GBM-derived exosomes loaded with selumetinib had a specific antitumor effect on U87MG cells and were non-toxic to normal brain cells. These exosomes offer improved therapeutic prospects for glioblastoma therapy. [ABSTRACT FROM AUTHOR]

Exosomes belong to the class of extracellular vesicles of endocytic origin, which are regarded as a promising source of cancer biomarkers in liquid biopsy. As a result, an accurate, sensitive, and specific quantification of these nano-sized particles is of significant importance. Affinity-based approaches are recognized as the most valuable technique for exosome isolation and characterization. Indeed, Affibody biomolecules are a type of protein scaffold engineered with small size and enjoy the features of high thermal stability, affinity, and specificity. While the utilization of antibodies, aptamers, and other biologically active substances for exosome detection has been reported widely, there are no reports describing Affibody molecules' usage for exosome detection. In this study, for the first time, we have proposed a novel strategy of using Affibody functionalized microbeads (AffiBeads) for exosome detection with a high degree of efficiency. As a proof-of-concept, anti-EGFR-AffiBeads were fabricated and applied to capture and detect human lung A549 cancer cell-derived EGFR-positive exosomes using flow cytometry and fluorescent microscopy. Moreover, the capture efficiency of the AffiBeads were compared with its counterpart antibody. Our results showed that the Affibody probe had a detection limit of 15.6 ng exosomes per mL (~12 exosomes per AffiBead). The approach proposed in the current study can be used for sensitive detection of low expression level markers on tumor-derived exosomes, providing a basis for early-stage cancer diagnosis. [ABSTRACT FROM AUTHOR]