Minimum information to report about a flow cytometry experiment on extracellular vesicles: Communication from the ISTH SSC subcommittee on vascular biology

The Extracellular Vesicle Flow Cytometry Working Group ( http://www.evflowcytometry.org ) is formed by members of the International Society for Extracellular Vesicles (ISEV), the International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH). This working group of flow cytometry experts develops guidelines for best practices regarding flow cytometry detection of extracellular vesicles. To improve rigor and standardization, this working group published a framework outlining the minimal information to report about a flow cytometry experiment on extracellular vesicles (MIFlowCyt-EV) in the Journal of Extracellular Vesicles, the ISEV journal, in 2020. In parallel, an article explaining MIFlowCyt-EV was published in Cytometry Part A, one of the ISAC journals, and now will be introduced to the ISTH as an SSC Communication in the Journal of Thrombosis and Haemostasis. The goal of this SSC Communication is to explain why flow cytometry is becoming the instrument of choice to characterize single extracellular vesicles, the obstacles that have been identified and (mostly) overcome by developing procedures to calibrate flow cytometers, and the relevance of reporting minimal information to improve reliability and reproducibility of experiments in which flow cytometers are used for characterization of extracellular vesicles.     

Anomaly of cornea and ocular adnexa in spinster homolog 2 (Spns2) knockout mice

Spinster 2 (Spns2) is a transporter that pumps sphingosine-1-phosphate (S1P), a bioactive lipid mediator synthesized in the cytoplasm, out of cells into the inter cellular space. S1P is a signal that modulates cellular behavior during embryonic development, inflammation and tissue repair, etc. A Spns2-null (KO) mouse is born with failure of eyelid closure (eyelid-open-at birth; EOB) and develop corneal fibrosis in adulthood. It remains elusive whether corneal lesion is caused by exposure to keratitis (lagophthalmos) of EOB phenotype or the loss of Spns2 directly perturbs the corneal tissue morphogenesis and intra-eyelid structures. Therefore, we investigated differences between the cornea and ocular adnexa morphogenesis in KO and wild-type (WT) embryos and adults as well.The loss of Spns2 perturbs cornea morphogenesis during embryonic development as early as E16.5 besides EOB phenotype. Histology showed that the corneal stroma was thinner with less extracellular matrix accumulation, e.g., collagen and keratocan in the KO mouse. Epithelial stratification, expression of keratin 12 and formation of desmosomes and hemidesmosomes were also perturbed in these KO corneas. Lacking Spns2 impaired morphogenesis of the Meibomian glands and of orbicularis oculi muscles. KO glands were labeled for ELOVL4 and PPARγ and were Oil-Red O-positive, suggesting KO acinar cells possessed functionality as the glands.This is the first report on the roles of Spns2 in corneal and Meibomian gland morphogenesis. Corneal tissue destruction in an adult KO mouse might be due to not only lagophthalmos but also to an impaired morphogenesis of cornea, Meibomian glands, and orbicularis oculi muscle.     

uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells

Malignant melanoma is a highly aggressive skin cancer characterized by an elevated grade of tumor cell plasticity. Such plasticity allows adaptation of melanoma cells to different hostile conditions and guarantees tumor survival and disease progression, including aggressive features such as drug resistance. Indeed, almost 50% of melanoma rapidly develop resistance to the BRAFV600E inhibitor vemurafenib, with fast tumor dissemination, a devastating consequence for patients’ outcomes. Vasculogenic mimicry (VM), the ability of cancer cells to organize themselves in perfused vascular-like channels, might sustain tumor spread by providing vemurafenibresistant cancer cells with supplementary ways to enter into circulation and disseminate. Thus, this research aims to determine if vemurafenib resistance goes with the acquisition of VM ability by aggressive melanoma cells, and identify a driving molecule for both vemurafenib resistance and VM. We used two independent experimental models of drug-resistant melanoma cells, the first one represented by a chronic adaptation of melanoma cells to extracellular acidosis, known to drive a particularly aggressive and vemurafenib-resistant phenotype, the second one generated with chronic vemurafenib exposure. By performing in vitro tube formation assay and evaluating the expression levels of the VM markers EphA2 and VE-cadherin by Western blotting and flow cytometer analyses, we demonstrated that vemurafenib-resistant cells obtained by both models are characterized by an increased ability to perform VM. Moreover, by exploiting the CRISPR-Cas9 technique and using the urokinase plasminogen activator receptor (uPAR) inhibitor M25, we identified uPAR as a driver of VM expressed by vemurafenib-resistant melanoma cells. Thus, uPAR targeting may be successfully leveraged as a new complementary therapy to inhibit VM in drug-resistant melanoma patients, to counteract the rapid progression and dissemination of the disease.     

Extracellular vesicles transmitted miR-31-5p promotes sorafenib resistance by targeting MLH1 in renal cell carcinoma

Sorafenib provides survival benefits in patients with advanced renal cell carcinoma (RCC), but its use is hampered by acquired drug resistance. It is important to fully clarify the molecular mechanisms of sorafenib resistance, which can help to avoid, delay or reverse drug resistance. Extracellular vesicles (EVs) can mediate intercellular communication by delivering effector molecules between cells. Here, we studied whether EVs are involved in sorafenib resistance of RCC and its possible molecular mechanisms. Using differential centrifugation, EVs were isolated from established sorafenib-resistant RCC cells (786-0 and ACHN), and EVs derived from sorafenib-resistant cells were uptaken by sensitive parental RCC cells and thus promoted drug resistance. Elevated exogenous miR-31-5p within EVs effectively downregulated MutL homolog 1 (MLH1) expression and thus promoted sorafenib resistance in vitro. Mice experiments also confirmed that miR-31-5p could mediate drug sensitivity in vivo. In addition, low expression of MLH1 was observed in sorafenib-resistant RCC cells and upregulation of MLH1 expression restored the sensitivity of resistant cell lines to sorafenib. Finally, miR-31-5p level in circulating EVs of RCC patients with progressive disease (PD) during sorafenib therapy was higher when compared to that in the pretherapy status. In conclusion, EVs shuttled miR-31-5p can transfer resistance information from sorafenib-resistant cells to sensitive cells by directly targeting MLH1, and thus magnify the drug resistance information to the whole tumor. Furthermore, miR-31-5p and MLH1 could be promising predictive biomarkers and therapeutic targets to prevent sorafenib resistance.     

Circulating MicroRNAs and Extracellular Vesicle–Containing MicroRNAs as Response Biomarkers of Anti–programmed Cell Death Protein 1 or Programmed Death-Ligand 1 Therapy in NSCLC

IntroductionAnti–programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) antibody therapy is a standard treatment for advanced NSCLC, and PD-L1 immunohistochemistry is used as a predictive biomarker for therapeutic response. However, because not all patients with NSCLC with high PD-L1 respond, and some patients with low PD-L1 expression exhibit durable benefit, more accurate predictive biomarkers are needed. Circulating microRNA (miRNA) and miRNA packaged in extracellular vesicles (EVs) are believed to play a role in intercellular communication among immune cells and between immune cells and tumor cells and may represent a good source of mechanism-related biomarkers.MethodsPretreatment plasma of patients with advanced NSCLC treated with single-agent anti–PD-1 or anti–PD-L1 antibody was used in this study. Plasma EVs were isolated using size-exclusion chromatography. Whole plasma and EV-containing RNAs were extracted. The miRNA profile was analyzed with a next-generation sequencing platform.ResultsSamples from 14 responders (patients who exhibited partial response or stable disease ≥6 mo) and 15 nonresponders (patients who exhibited progressive disease as per Response Evaluation Criteria in Solid Tumors) were analyzed. In total, 32 miRNAs (p = 0.0030–0.0495) from whole plasma and seven EV-associated miRNAs (p = 0.041–0.0457) exhibited significant concentration differences between responders and nonresponders. The results of some of these circulating miRNAs were validated in a separate cohort with eight responders and 13 nonresponders. The tumor PD-L1 level was also assessed using immunohistochemistry for patients involved in both cohorts.ConclusionsSpecific circulating miRNAs in whole plasma and plasma EVs are differentially expressed between responders and nonresponders and have potential as predictive biomarkers for anti–PD-1/PD-L1 treatment response.     

Inhibition of PI3K/Akt/NF-κB signaling by Aloin for ameliorating the progression of osteoarthritis: In vitro and in vivo studies

Osteoarthritis (OA) is a progressive and degenerative joint disease. Aloin is a bitter and yellow-brown-coloured compound from the Aloe plant and is allowed for use in foods as a “natural flavour”. In our study, we examined the protective effects of Aloin on the inhibition of OA development as well as its underlying mechanism in both in vitro and vivo experiments. In in-vitro experiments, the protective effect of aloin on the anabolism and catabolism of the extracellular matrix (ECM) induced by IL-1 β in chondrocytes by inhibiting the expression of pro-inflammatory factors, including TNF-α (p = 0.016), IL-6 (p = 0.006), iNOS (p = 0.001) and COX-2 (p = 0.006). Mechanistically, Aloin suppressed the IL-1β-induced activation of the PI3K/Akt/NF-κB signalling pathway cascades. Moreover, molecular docking studies demonstrated that Aloin bound strongly to PI3K. In vivo, Aloin ameliorated the OA process in the destabilization of the medial meniscus (DMM) model.In summary, our findings demonstrate that Aloin ameliorates the progression of OA via the PI3K/Akt/NF-κB signalling pathways, which supports Aloin as a promising therapeutic agent for the treatment of OA.     

Induction of specific cell‐mediated immune responses and protection in BALB/c mice by vaccination with outer membrane vesicles from a Brucella melitensis human isolate

Brucellosis is a worldwide bacterial zoonosis caused by Brucella spp. No approved vaccine is available for human use against the disease. In this study, outer membrane vesicles (OMVs) from a Brucella melitensis biovar 1 human isolate obtained in Iran were used to immunize BALB/c mice (n = 12) by 2 intramuscular injections with a 2‐week interval. Another group of 12 mice was used as non‐vaccinated controls. Two weeks after the last vaccination, six mice of each group were sacrificed, and proliferation and interferon gamma (IFNγ) production responses of their splenocytes were evaluated following in vitro stimulation with killed Brucella cells. The other mice were challenged with the virulent B. melitensis isolate. Two weeks later, mice were killed and spleens were cultured to determine the number of the challenge strain. The results showed proliferative response and IFNγ production of splenocytes from vaccinated mice (stimulation index: 2.18 ± 0.57, and 1519.35 ± 10.70 pg/mL, respectively) were significantly higher than those of control mice (stimulation index: 1.02 ± 0.02, and 210.01 ± 17.58 pg/mL, respectively). Numbers of the challenge strain in spleens of vaccinated mice were also significantly less than those in the controls with 1.6 units of protection. Our study revealed vaccination with OMVs of the B. melitensis isolate could induce specific immune responses and protection against infection in the mouse model suggesting their potential application for active immunization against brucellosis.     

Cancer Stem Cells and Circulatory Tumor Cells Promote Breast Cancer Metastasis

Breast cancer (BC) is a highly metastatic, pathological cancer that significantly affects women worldwide. The mortality rate of BC is related to its heterogeneity, aggressive phenotype, and metastasis. Recent studies have highlighted that the tumor microenvironment (TME) is critical for the interplay between metastasis mediators in BC. BC stem cells, tumor-derived exosomes, circulatory tumor cells (CTCs), and signaling pathways dynamically remodel the TME and promote metastasis. This review examines the cellular and molecular mechanisms governing the epithelial to mesenchymal transition (EMT) that facilitate metastasis. This review also discusses the role of cancer stem cells (CSCs), tumor-derived exosomes, and CTs in promoting BC metastasis. Furthermore, the review emphasizes major signaling pathways that mediate metastasis in BC. Finally, the interplay among CSCs, exosomes, and CTCs in mediating metastasis have been highlighted. Therefore, understanding the molecular cues that mediate the association of CSCs, exosomes, and CTCs in TME helps to optimize systemic therapy to target metastatic BC.     

Fibroageing: An ageing pathological feature driven by dysregulated extracellular matrix-cell mechanobiology

Ageing is a multifactorial biological process leading to a progressive decline of physiological functions. The process of ageing includes numerous changes in the cells and the interactions between cell-cell and cell-microenvironment remaining as a critical risk factor for the development of chronic degenerative diseases. Systemic inflammation, known as inflammageing, increases as a consequence of ageing contributing to age-related morbidities. But also, persistent and uncontrolled activation of fibrotic pathways, with excessive accumulation of extracellular matrix (ECM) and organ dysfunction is markedly more frequent in the elderly. In this context, we introduce here the concept of Fibroageing, that is, the propensity to develop tissue fibrosis associated with ageing, and propose that ECM is a key player underlying this process. During ageing, molecules of the ECM become damaged through many modifications including glycation, crosslinking, and accumulation, leading to matrix stiffness which intensifies ageing-associated alterations. We provide a framework with some mechanistic hypotheses proposing that stiff ECM, in addition to the well-known activation of fibrotic positive feedback loops, affect several of the hallmarks of ageing, such as cell senescence and mitochondrial dysfunction, and in this context, is a key mechanism and a driver thread of Fibroageing.