Journal of Extracellular Vesicles

A Predictive Model for Initial Platinum‐Based Chemotherapy Efficacy in Patients with Postoperative Epithelial Ovarian Cancer Using Tissue‐Derived Small Extracellular Vesicles

AbstractEpithelial ovarian cancer (EOC) is an often‐fatal malignancy marked by the development of resistance to platinum‐based chemotherapy. Thus, accurate prediction of platinum drug efficacy is crucial for strategically selecting postoperative interventions to mitigate the risks associated with suboptimal therapeutic outcomes and adverse effects. Tissue‐derived extracellular vesicles (tsEVs), in contrast to their plasma counterparts, have emerged as a powerful tool for examining distinctive attributes of EOC tissues. In this study, 4D data‐independent acquisition (DIA) proteomic sequencing was performed on tsEVs obtained from 58 platinum‐sensitive and 30 platinum‐resistant patients with EOC. The analysis revealed a notable enrichment of differentially expressed proteins that were predominantly associated with immune‐related pathways. Moreover, pivotal immune‐related proteins (IRPs) were identified by LASSO regression. These factors, combined with clinical parameters selected through univariate logistic regression, were used for the construction of a model employing multivariate logistic regression. This model integrated three tsEV IRPs, CCR1, IGHV_35 and CD72, with one clinical parameter, the presence of postoperative residual lesions. Thus, this model could predict the efficacy of initial platinum‐based chemotherapy in patients with EOC post‐surgery, providing prognostic insights even before the initiation of chemotherapy.

Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer

AbstractHigh‐grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients’ ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of ‘core ascitic EV‐associated proteins’ but also defined their subset unique to HGSC ascites. Using single‐cell RNA sequencing data, we mapped the origin of HGSC‐specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non‐malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type‐specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics.

Cisplatin Promotes Immunosuppression in Ovarian Cancer by Enhancing miR‐181a‐5p‐Enriched Extracellular Vesicles to Drive Regulatory T Cell Differentiation

ABSTRACT Although cisplatin (CDDP) is widely employed in combination immunotherapy, no CDDP‐based regimen has shown a survival benefit when treating the ovarian cancers. This is mainly because the impact of CDDP on immunotherapy is not fully understood. A critical gap concerns how CDDP reshape the tumour microenvironment, especially through extracellular vesicles (EVs)‐mediated communication. In this work, using human and murine ovarian cancer cells as a model, we demonstrate that CDDP boosts the secretion of EVs from cancer cells, while exerting no such effect on non‐cancerous cells. These CDDP‐induced tumour‐derived EVs, in turn, drive the differentiation of CD4 + T cells towards immunosuppressive regulatory T cells (T reg cells), which are known to limit the efficacy of immunotherapy. Based on next‐generation sequencing, a significant enrichment of miR‐181a‐5p was identified in CDDP‐induced tumour‐derived EVs, and further functional studies confirmed that this microRNA promoted T reg cell differentiation via suppressing sirtuin 1 (SIRT1), a key regulator of the transcription factor forkhead box protein P3 (FOXP3). Importantly, inhibition of miR‐181a‐5p abrogated the T reg ‐promoting effect of CDDP‐induced tumour‐derived EVs, a finding further validated in vivo , where blockade of miR‐181a‐5p not only impaired T reg differentiation but also restored T‐cell‐mediated antitumour immunity and restrained tumour growth. Together, these findings uncover a previously unrecognised mechanism by which CDDP exacerbates immunosuppression via miR‐181a‐5p‐enriched EVs and suggest that targeting this pathway could improve the therapeutic efficacy of combination immunotherapy in ovarian cancer.

Small extracellular vesicles ameliorate peripheral neuropathy and enhance chemotherapy of oxaliplatin on ovarian cancer

AbstractThere are no effective treatments for chemotherapy induced peripheral neuropathy (CIPN). Small extracellular vesicles (sEVs) facilitate intercellular communication and mediate nerve function and tumour progression. We found that the treatment of mice bearing ovarian tumour with sEVs derived from cerebral endothelial cells (CEC‐sEVs) in combination with a chemo‐drug, oxaliplatin, robustly reduced oxaliplatin‐induced CIPN by decreasing oxaliplatin‐damaged myelination and nerve fibres of the sciatic nerve and significantly amplified chemotherapy of oxaliplatin by reducing tumour size. The combination therapy substantially increased a set of sEV cargo‐enriched miRNAs, but significantly reduced oxaliplatin‐increased proteins in the sciatic nerve and tumour tissues. Bioinformatics analysis revealed the altered miRNAs and proteins formed two distinct networks that regulate neuropathy and tumour growth, respectively. Intravenously administered CEC‐sEVs were internalized by axons of the sciatic nerve and cancer cells. Reduction of CEC‐sEV cargo miRNAs abolished the effects of CEC‐sEVs on oxaliplatin‐inhibited axonal growth and on amplification of the anti‐cancer effect in ovarian cancer cells, suggesting that alterations in the networks of miRNAs and proteins in recipient cells contribute to the therapeutic effect of CEC‐sEVs on CIPN. Together, the present study demonstrates that CEC‐sEVs suppressed CIPN and enhanced chemotherapy of oxaliplatin in the mouse bearing ovarian tumour.

Chemotherapy‐induced release of ADAM17 bearing EV as a potential resistance mechanism in ovarian cancer

AbstractOvarian cancer (OvCa) is the gynaecological disorder with the poorest prognosis due to the fast development of chemoresistance. We sought to connect chemoresistance and cancer cell‐derived extracellular vesicles (EV). The mechanisms of how chemoresistance is sustained by EV remained elusive. One potentially contributing factor is A Disintegrin and Metalloprotease 17 (ADAM17)—itself being able to promote chemoresistance and inducing tumour cell proliferation and survival via the Epidermal Growth Factor Receptor (EGFR) pathway by shedding several of its ligands including Amphiregulin (AREG). We now demonstrate that upon chemotherapeutic treatment, proteolytically active ADAM17 is released in association with EV from OvCa cells. In terms of function, we show that patient‐derived EV induce AREG shedding and restore chemoresistance in ADAM17‐deficient cells. Confirming that ADAM17‐containing EV transmit chemoresistance in OvCa, we propose that ADAM17 levels (also on EV) might serve as an indicator for tumour progression and the chemosensitivity status of a given patient.

Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions

AbstractSeparating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size‐exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context‐dependent and time‐dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context‐dependent and/or time‐dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.

Antibody‐Targeted Artificial T Cell and Natural Killer Cell Derived Vesicles for Cancer Immunotherapy

ABSTRACT T cell and NK92 cell‐derived extracellular vesicles or artificial cell‐derived vesicles (ACDVs) exhibit anti‐cancer effects through inherited cytotoxic proteins like perforin and granzyme or transmembrane receptors like FasL and TRAIL. The anti‐cancer potential of artificial T and NK vesicles has been improved by attaching targeting moieties to their surface using genetic engineering or covalent surface modifications. However, the genetic engineering of immune cells from which the vesicles are derived is laborious, expensive, and inefficient, and naturally derived exosomes are released in low quantities. Here, we compared the properties of tumour‐targeted and untargeted ACDVs from activated‐T cells and NK92 cells. We examined whether the cancer cell‐killing capacity of ACDVs derived from activated‐T and NK92 cells could be targeted to cancer cells by conjugating tumour‐targeting antibodies to their surface. We targeted T and NK92 ACDVs to cancer cells possessing the xenoantigen, N‐glycolyl neuraminic acid GM3 ganglioside, using the 14f7hT antibody or the tumour antigen, epidermal growth factor receptor, using the nimotuzumab antibody. Antibody targeting improved the cell interaction, internalization, and cytotoxicity of T and NK92 ACDVs. Interestingly, the T‐ACDVs retained perforin, granzyme, FasL and TRAIL, whereas NK92 ACDVs retained perforin, granzyme and FasL. Based on their ease of production and lower cost, we chose NK92 ACDVs for in vivo and ex vivo studies. Intravenously injected nimotuzumab‐conjugated NK92 ACDVs decreased the tumour volumes of EGFR‐expressing ovarian cancer xenografts in mice. 14F7hT‐conjugated NK92 ACDVs showed cytotoxic activity against chronic lymphocytic leukaemia biopsies. This research shows the potential for using antibody‐conjugated, cytotoxic T and NK ACDVs as a feasible and effective approach for tumour‐targeted immunotherapy.