In patients with prostate cancer (PCa), bone lesions appear osteoblastic in radiographs; however, pathological fractures frequently occur in PCa patients, and bone resorption is observed in all metastatic lesions under histopathologic assessment. The mechanisms that balance the activities of osteoblasts and osteoclasts in PCa patients remain unclear. We unexpectedly discovered that PCa exosomes are critical mediators in the regulation of bone homeostasis that results in osteoclastic lesions and thereby promotes tumor growth in bone. We evaluated how exosomes derived from osteoblastic, osteoclastic, and mixed PCa cell lines affect osteoblast and osteoclast differentiation, revealing that all three types of PCa exosomes promoted osteoclastogenesis in vitro and induced osteolysis in vivo. Mechanistically, microRNAs (miRNAs) delivered by PCa exosomes were found to play several key roles in bone homeostasis. Among the delivered miRNAs, miR‐92a‐1‐5p, the most abundant miRNA, downregulated type I collagen expression by directly targeting COL1A1, and thus promoting osteoclast differentiation and inhibiting osteoblastogenesis. Furthermore, PCa exosomes also markedly reduced type I collagen expression in vivo. Our findings not only offer a novel perspective on tumor bone metastasis, where—contrary to our initial hypothesis—exosomes derived from an osteoblastic tumor induce osteoclast differentiation, but also suggest potential therapeutic targets for PCa bone metastasis.

Extracellular vesicles (EVs) are taken up by most cells, however specific or preferential cell targeting remains a hurdle. This study aims to develop an EV that targets cells involved in inflammation, specifically those expressing intercellular adhesion molecule-1 (ICAM-1). To target these cells, we overexpress the ICAM-1 binding receptor “lymphocyte function-associated antigen-1” (LFA-1) in HEK293F cells, by sequential transfection of plasmids of the two LFA-1 subunits, ITGAL and ITGB2 (CD11a and CD18). The LFA-1 receptor was strongly overexpressed on the EVs released by the transfected cells. We further loaded these EVs with a therapeutic peptide, targeting myeloid differentiation primary response 88 (Myd88; EV^Myd88), through a developed EV open-and-close procedure. Myd88 is a downstream common intracellular messenger for most TLR-receptors. EV expression of LFA-1 increases EV binding to ICAM-1-expressing cells, an effect that was dose-dependently inhibited by a specific neutralizing ICAM-1 antibody. Further, activated human endothelial cells treated with LFA-1 EV^Myd88 had increased uptake of these EVs, resulting in dose-dependent inhibition of induced release of IL-8, presumably by targeting Myd88. We conclude that LFA-1-expressing EV^Myd88 may be a candidate suitable for delivering therapeutic peptides in inflammatory diseases associated with TLR-activation. Graphical Abstract

Background: We have previously reported that extracellular vesicles (EVs) derived from osteoblastic, osteoclastic and mixed prostate cancer cells promote osteoclast differentiation and inhibit osteoblast differentiation via transferring miR-92a-1-5p. In the present study, we focused on engineering miR-92a-1-5p into EVs and determining any therapeutic roles and mechanisms of the engineered EVs. Methods: A stable prostate cancer cell line (MDA PCa 2b) overexpressing miR-92a-1-5p was constructed by lentivirus, and EVs were isolated by ultracentrifugation. The overexpression of miR-92a-1-5p in both cells and EVs was tested using qPCR. Osteoclast function was evaluated by Trap staining, mRNA expression of osteoclastic markers ctsk and trap, immunolabeling of CTSK and TRAP and microCT using either in vitro and in vivo assays. Target gene of miR-92a-1-5p was proved by a dual-luciferase reporter assay system. siRNAs were designed and used for transient expression in order to determine the role of downstream genes on osteoclast differentiation. Results: Stable overexpression cells of miRNA-92a-5p was associated with EVs upregulating this microRNA, as confirmed by qPCR. Further, miR-92a-1-5p enriched EVs promote osteoclast differentiation in vitro by reducing MAPK1 and FoxO1 expression, associated with increased osteoclast function as shown by TRAP staining and mRNA expression of osteoclast functional genes. siRNA targeting MAPK1 or FoxO1 resulted in similar increase in osteoclast function. In vivo, the miR-92a-1-5p enriched EVs given via i.v. injection promote osteolysis, which was associated with reduction of MAPK1 and FoxO1 expression in bone marrow. Conclusion: These experiments suggest that miR-92a-1-5p enriched EVs regulate osteoclast function via reduction of MAPK1 and FoxO1. Graphical

Androgen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer (PCa). However, after 2–3 years ADT treatment, prostate cancer inevitably transits from androgen-dependent PCa (ADPC) to androgen-independent PCa (AIPC), which has a poor prognosis owing to its unclear mechanism and lack of effective therapeutic targets. Small extracellular vesicles (sEVs) play a vital role in the development of cancer. However, the role of PCa sEVs in the transformation of AIPC remains poorly understood. Two different cell models were employed and compared. sEVs from ADPC cells (LNCaP) and AIPC cells (LNCaP-AI + F cells) were isolated and characterized. After co-culture of LNCaP-AI + F sEVs with LNCaP cells and of LNCaP sEVs with LNCaP-AI + F cells, androgen-independent transformation was determined respectively. Mechanically, small RNA sequencing was performed. Androgen-independent transformation was examined by the upregulation and downregulation of miRNA and downstream pathways were analyzed. LNCaP-AI + F sEVs promoted the androgen-independent transformation of LNCaP cells. Interestingly, LNCaP sEVs exhibited a capacity to reverse the process.Let-7a-5p transfer was demonstrated. Furthermore, let-7a-5p overexpression promotes the androgen-independent transformation and let-7a-5p down-regulation reverses the process. Androgen receptor (AR) and PI3K/Akt pathways were identified and demonstrated by both let-7a-5p regulation and PCa sEVs coculture. PCa sEVs are intimately involved in the regulation of androgen-independent transformation of prostate cancer by transferring the key sEVs molecular let-7a-5p and then activating the AR and PI3K/Akt signaling pathways. Our results provide new perspectives for the development of sEVs and sEVs molecular targeted treatment approaches for AIPC patients.

Prostate cancer (PCa) is a leading male malignancy worldwide, with metabolic reprogramming being a critical hallmark of its progression. Extracellular vesicles (EVs) derived from tissues directly reflect the tumor microenvironment, offering unique insights into cancer pathophysiology that are unattainable through cell line or biofluid‐derived EVs. However, the functional roles of tissue‐derived EVs in PCa metabolism remain poorly understood. Leveraging our expertise in murine PCa model establishment and EV isolation from prostate tissue, this study aimed to characterize functional differences between PCa and normal prostate tissue via proteomic analysis of tissue‐derived sEVs. We orthotopically implanted luciferase‐labeled PCa cells into nude mice to establish an in situ PCa model, confirmed tumor formation via in vivo imaging, and harvested tissues after 4 weeks. sEVs were isolated using ultracentrifugation combined with an iodixanol density cushion and characterized by transmission electron microscopy, nanoparticle tracking analysis, and protein marker profiling. Proteomic analysis identified 28 upregulated and 24 downregulated proteins in PCa‐derived sEVs compared to normal controls. Subcellular localization revealed enrichment in the cytoplasm, while pathway analysis highlighted significant involvement in metabolic processes, particularly glycolysis, amino acid biogenesis, carbon metabolism, and pyruvate metabolism. Our study establishes a robust method for isolating prostate tissue sEVs and provides the first evidence that PCa tissue‐derived sEVs exhibit profound metabolic pathway alterations. These findings enhance our understanding of PCa progression mechanisms and may facilitate the development of novel diagnostic biomarkers and therapeutic strategies targeting metabolic dysregulation in PCa. Summary: In this study, we created a method to isolate prostate tissue small EVs, based on our knowledge of the murine prostate cancer model building. Our data suggested that prostate tissue small EVs proteins significantly changed in many metabolism pathways, such as Glycolysis, Biogenesis of amino acids, Carbon metabolism and Pyruvate metabolism. In this study, we are the first to report prostate tissue‐derived EVs proteins enriched in alterations of cancer metabolism. These differential proteins in PCa tissue EVs reflect metabolic changes in PCa and may provide insights into the development of early diagnostic biomarkers or novel therapeutic strategies.