Tetraspanin CD9 Research Articles

Lack of involvement of CD63 and CD9 tetraspanins in the extracellular vesicle content delivery process

Extracellular vesicles (EVs) are thought to mediate intercellular communication by transferring cargoes from donor to acceptor cells. The EV content-delivery process within acceptor cells is still poorly characterized and debated. CD63 and CD9, members of the tetraspanin family, are highly enriched within EV membranes and are respectively enriched within multivesicular bodies/endosomes and at the plasma membrane of the cells. CD63 and CD9 have been suspected to regulate the EV uptake and delivery process. Here we used two independent assays and different cell models (HeLa, MDA-MB-231 and HEK293T cells) to assess the putative role of CD63 and CD9 in the EV delivery process that includes uptake and cargo delivery. Our results suggest that neither CD63, nor CD9 are required for this function.

La orina más allá de los electrolitos: diagnóstico a través de las vesículas extracelulares

Background and objectiveExtracellular vesicles (EVs) reflect the pathophysiological state of their cells of origin and are a reservoir of renal information accessible in urine. When biopsy is not an option, EVs present themselves as sentinels of function and damage, providing a non-invasive approach. However, the analysis of EVs in urine requires prior isolation, which slows down and hinders translation to clinical practice. The aim of this study is to show the applicability of the “single particle interferometric reflectance imaging sensor” (SP-IRIS) technology through the ExoView® platform for the direct analysis of urine EVs and proteins involved in renal function. Materials and methodsThe ExoView® technology enables the quantification and phenotyping of EVs present in urine and the quantification of their membrane and internal proteins. We have applied this technology to the quantification of urinary EVs and their proteins with renal tubular expression, amnionless (AMN) and secreted frizzled-related protein 1 (SFRP1), using only 5μl of urine. Tubular expression was confirmed by immunohistochemistry. ResultsThe mean size of the EVs analysed was 59±16nm for those captured by tetraspanin CD63, 61±16nm for those captured by tetraspanin CD81, and 59±10 nm for tetraspanin CD9, with CD63 being the majority EVs subpopulation in urine (48.92%). The distribution of AMN and SFRP1 in the three capture tetraspanins turned out to be similar for both proteins, being expressed mainly in CD63 (48.23% for AMN and 52.1% for SFRP1). ConclusionsThis work demonstrates the applicability and advantages of the ExoView® technique for the direct analysis of urine EVs and their protein content in relation to the renal tubule. The use of minimum volumes, 5μl, and the total analysis time not exceeding three hours facilitate the translation of EVs to daily clinical practice as source of diagnostic information.

Ontogenesis of functional platelet subpopulations from preterm and term neonates to adulthood: The PLINIUS study

AbstractErythrocytes undergo a well-defined switch from fetal to postnatal circulation, which is mainly reflected by the stage-specific expression of hemoglobin chains. Perinatal alterations in thrombopoiesis are poorly understood. We assessed the ontogenesis of platelet phenotype and function from early prematurity to adulthood. We recruited 64 subjects comprising 7 extremely preterm (27-31 weeks gestational age), 25 moderately preterm (32-36 weeks), 10 term neonates, 8 infants (<2 years), 5 children (2-13 years), and 9 adults (>13 years). Blood was withdrawn at up to 3 different time points in neonates (t1: 0-2, t2: 3-7, and t3: 8-14 days after birth). We found that the expression levels of the major surface receptors for fibrinogen, collagen, vWF, fibronectin, and laminin were reduced but correlated with decreased platelet size, indicating a normal surface density. Although CD62P and CD63 surface exposure upon stimulation with TRAP-6, ADP, or U46619 was unaltered or only slightly reduced in neonates, GPIIb/IIIa inside-out and outside-in activation was blunted but showed a continuous increase until adulthood, correlating with the expression of the GPIIb/IIIa regulating tetraspanin CD151. Platelet subpopulation analysis using automated clustering revealed that neonates presented with a CD63+/PAC-1– pattern, followed by a continuous increase in CD63+/PAC-1+ platelets until adulthood. Our findings revealed that the number of platelet-monocyte and platelet-neutrophil aggregates, but not platelet-lymphocyte aggregates, is increased in neonates and that neonatal aggregate formation depends in part on CD62P activation. Our PLatelets In Neonatal Infants Study (PLINIUS) provides several lines of evidence that the platelet phenotype and function evolve continuously from neonates to adulthood.

Targeting of Tetraspanin CD81 with Monoclonal Antibodies and Small Molecules to Combat Cancers and Viral Diseases

Tetraspanin CD81 plays major roles in cell-cell interactions and the regulation of cellular trafficking. This cholesterol-embarking transmembrane protein is a co-receptor for several viruses, including HCV, HIV-1 and Chikungunya virus, which exploits the large extracellular loop EC2 for cell entry. CD81 is also an anticancer target implicated in cancer cell proliferation and mobility, and in tumor metastasis. CD81 signaling contributes to the development of solid tumors (notably colorectal, liver and gastric cancers) and has been implicated in the aggressivity of B-cell lymphomas. A variety of protein partners can interact with CD81, either to regulate attachment and uptake of viruses (HCV E2, claudin-1, IFIM1) or to contribute to tumor growth and dissemination (CD19, CD44, EWI-2). CD81-protein interactions can be modulated with molecules targeting the extracellular domain of CD81, investigated as antiviral and/or anticancer agents. Several monoclonal antibodies anti-CD81 have been developed, notably mAb 5A6 active against invasion and metastasis of triple-negative breast cancer cells. CD81-EC2 can also be targeted with natural products (trachelogenin and harzianoic acids A-B) and synthetic compounds (such as benzothiazole-quinoline derivatives). They are weak CD81 binders but offer templates for the design of new compounds targeting the open EC2 loop. There is no anti-CD81 compound in clinical development at present, but this structurally well-characterized tetraspanin warrants more substantial considerations as a drug target.

Plasma‑derived CD16 exosomes and peripheral blood monocytes as correlating biomarkers in head and neck cancer.

Exosomes play an important role in the individual immune regulation in patients with head and neck squamous cell carcinoma (HNSCC) as part of the tumor microenvironment. Patients with HNSCC with advanced tumor stages reveal significantly increased levels of plasma derived CD16+ (FcRIIIA) total exosomes as demonstrated in our previous study. Furthermore, increased individual abundances of peripheral blood CD16+ non-classical monocytes have been shown to correlate with increased monocytic programmed death ligand 1 (PD-L1) and CD4+ T cell disturbances in oropharyngeal cancer. However, the context of plasma derived CD16+ exosomes in patients with HNSCC and their role in the immune-regulation of circulating monocyte subsets has not been investigated so far. In the present study, exosomes were isolated from plasma samples of healthy donors and patients with HNSCC and evaluated for their morphology, size and protein composition using transmission electron microscopy, western blotting and bead-based flow cytometry. Monocyte subset abundances were analyzed in whole blood measurements in terms of the CD14/CD16 cell surface expression patterns, different monocytic adhesion molecules and checkpoint molecule PD-L1 using flow cytometry. Isolated exosomes were positive for the tetraspanins CD63 and CD9 as well as the endosomal marker TSG101, but negative for the non-exosomal marker glucose-regulated protein 94 and apolipoprotein ApoA1. Plasma derived CD16+ exosomes and exosome size distribution were significantly correlated with abundances of CD16+ non-classical monocytes and CD16+ intermediate monocytes, respectively. Furthermore, the data revealed significant correlations between CD16+ plasma derived exosomes and adhesion molecules CD29 (integrin β1) and CX3CR1 on certain monocyte subsets. These data suggested that CD16 positive exosomes and exosome size distribution were potential surrogates to characterize the composition of monocyte subsets in patients with HNSCC. Overall, both CD16-positive exosomes and CD16-positive monocyte subsets are potential liquid biomarkers that could be used to characterize the individual immunological situation of patients with HNSCC.

Abstract 1349: Exploring CD63 as a targetable mediator of the pro-tumorigenic tissue microenvironment in HNSCC

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) is one of the ten most common cancer types worldwide - ranked on the sixth position with 560,000 new diagnoses and 270,000 deaths in 2020. The survival could be increased only slightly over the past decades and in addition the incidence of HNSCC continues to rise. In this study, we investigated the role of the tetraspanin CD63 during cancer progression. Methods: First, we performed survival analyses with available statistics from data bases to correlate CD63 and survival. Then, we established a 20-marker spectral FACS panel to identify different fibroblast subpopulations in patient tissue. Upon receiving patient consent, fresh HNSCC tissue was received from surgical tumor resections at the University Hospital of Bonn. We mechanically and enzymatically digested and purified the tissue of ten patients to create single-cell suspensions. After staining, the cells were measured on a Sony ID7000 Spectral Analyzer and interpreted with FlowJo. To identify whether CD63 on fibroblasts affects the differentiation of macrophages in vitro, we isolated primary fibroblasts and monocytes from HNSCC patients and deleted CD63 in fibroblasts by CRISPR-Cas9. Thereafter, we co-incubated fibroblasts (without and with the CD63-Knockout) and macrophages over ten days in M-CSF containing medium, and measured the expression of M1 and M2 markers through flow cytometry. Results: First, correlating CD63 gene expression with the patient outcome showed that CD63 correlated with a decreased survival. Then, analyzing fresh tumor specimens with the multicolor panel showed an increase of CD63 expression in different fibroblast subpopulations in the tumor specimens, e. g. CD9+ CD34+ CD63+ CD90+ fibroblasts increased more than eightfold. Investigating potential mechanisms, by which CD63 expression on fibroblasts affects tumor progression, deleting CD63 steered macrophages towards the pro-inflammatory M1 phenotype. Conclusion: This study demonstrates that HNSCC increases CD63 expression in different fibroblast populations and that deleting CD63 on fibroblasts can steer macrophages towards the pro-inflammatory phenotype, thereby suggesting a rationale for testing CD63 as a therapeutic target. Citation Format: Elisabeth B. Dingendorf, Michael C. Stoffel, Dahlia L. Kittel, Glen Kristiansen, Tristan Lerbs. Exploring CD63 as a targetable mediator of the pro-tumorigenic tissue microenvironment in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1349.

Productive HIV-1 infection of tissue macrophages by fusion with infected CD4+ T cells.

Macrophages are essential for HIV-1 pathogenesis and represent major viral reservoirs. Therefore, it is critical to understand macrophage infection, especially in tissue macrophages, which are widely infected in vivo, but poorly permissive to cell-free infection. Although cell-to-cell transmission of HIV-1 is a determinant mode of macrophage infection in vivo, how HIV-1 transfers toward macrophages remains elusive. Here, we demonstrate that fusion of infected CD4+ T lymphocytes with human macrophages leads to their efficient and productive infection. Importantly, several tissue macrophage populations undergo this heterotypic cell fusion, including synovial, placental, lung alveolar, and tonsil macrophages. We also find that this mode of infection is modulated by the macrophage polarization state. This fusion process engages a specific short-lived adhesion structure and is controlled by the CD81 tetraspanin, which activates RhoA/ROCK-dependent actomyosin contractility in macrophages. Our study provides important insights into the mechanisms underlying infection of tissue-resident macrophages, and establishment of persistent cellular reservoirs in patients.

Prognostic value and multifaceted roles of tetraspanin CD9 in cancer.

CD9 is a crucial regulator of cell adhesion in the immune system and plays important physiological roles in hematopoiesis, blood coagulation or viral and bacterial infections. It is involved in the transendothelial migration of leukocytes which might also be hijacked by cancer cells during their invasion and metastasis. CD9 is found at the cell surface and the membrane of exosomes affecting cancer progression and therapy resistance. High expression of CD9 is mostly associated with good patients outcome, with a few exceptions. Discordant findings have been reported for breast, ovarian, melanoma, pancreatic and esophageal cancer, which might be related to using different antibodies or inherent cancer heterogeneity. According to in vitro and in vivo studies, tetraspanin CD9 is not clearly associated with either tumor suppression or promotion. Further mechanistic experiments will elucidate the role of CD9 in particular cancer types and specific conditions.

Single vesicle analysis reveals the release of tetraspanin positive extracellular vesicles into circulation with high intensity intermittent exercise.

Small extracellular vesicles (sEVs) are released from all cell types and participate in the intercellular exchange of proteins, lipids, metabolites and nucleic acids. Proteomic, flow cytometry and nanoparticle tracking analyses suggest sEVs are released into circulation with exercise. However, interpretation of these data may be influenced by sources of bias introduced by different analytical approaches. Seven healthy participants carried out a high intensity intermittent training (HIIT) cycle protocol consisting of 4×30s at a work-rate corresponding to 200% of individual max power (watts) interspersed by 4.5min of active recovery. EDTA-treated blood was collected before and immediately after the final effort. Platelet-poor (PPP) and platelet-free (PFP) plasma was derived by one or two centrifugal spins at 2500g, respectively (15min, room temperature). Platelets were counted on an automated haemocytometer. Plasma samples were assessed with the Exoview R100 platform, which immobilises sEVs expressing common tetraspanin markers CD9, CD63, CD81 and CD41a on microfluidic chips and with the aid of fluorescence imaging, counts their abundance at a single sEV resolution, importantly, without a pre-isolation step. There was a lower number of platelets in the PFP than PPP, which was associated with a lower number of CD9, CD63 and CD41a positive sEVs. HIIT induced an increase in fluorescence counts in CD9, CD63 and CD81 positive sEVs in both PPP and PFP. These data support the concept that sEVs are released into circulation with exercise. Furthermore, platelet-free plasma is the preferred, representative analyte to study sEV dynamics and phenotype during exercise. KEY POINTS: Small extracellular vesicles (sEV) are nano-sized particles containing protein, metabolites, lipid and RNA that can be transferred from cell to cell. Previous findings implicate that sEVs are released into circulation with exhaustive, aerobic exercise, but since there is no gold standard method to isolate sEVs, these findings may be subject to bias introduced by different approaches. Here, we use a novel method to immobilise and image sEVs, at single-vesicle resolution, to show sEVs are released into circulation with high intensity intermittent exercise. Since platelet depletion of plasma results in a reduction in sEVs, platelet-free plasma is the preferred analyte to examine sEV dynamics and phenotype in the context of exercise.

Screening of EWI-2-Derived Peptides for Targeting Tetraspanin CD81 and Their Effect on Cancer Cell Migration.

CD81, a transmembrane protein belonging to the tetraspanin family, has recently been suggested as a therapeutic target for cancers. Here, we screened peptides that bind to the tetraspanin CD81 protein, and evaluated their inhibitory activity in cancer cell migration. To screen for CD81-binding peptides (CD81-BP), a peptide array membrane was prepared from the amino acid sequence of the EWI-2 protein, a major partner of CD81, before binding to fluorescently labeled CD81. As a result, four candidate CD81-BPs were identified and characterized. In particular, the CFMKRLRK peptide (called P152 in this study) was found to be the best candidate that preferentially binds to the extracellular loop of CD81, with an estimated dissociation constant of 0.91 µM. Since CD81 was reported to promote cancer cell migration, an initial step in metastasis, the Boyden chamber assay, was next performed to assess the effect of CD81-BP candidates on the migration of MDA-MB-231 human breast cancer cells. Interestingly, our result indicated that P152 could suppress MDA-MB-231 cell migration at the level comparable to that of an anti-human CD81 antibody (5A6). Thus, we propose these CD81-BPs with the anti-migration property against cancer cells for the development of novel therapeutic strategies.

Extracellular vesicle distribution and localization in skeletal muscle at rest and following disuse atrophy

BackgroundSkeletal muscle (SkM) is a large, secretory organ that produces and releases myokines that can have autocrine, paracrine, and endocrine effects. Whether extracellular vesicles (EVs) also play a role in the SkM adaptive response and ability to communicate with other tissues is not well understood. The purpose of this study was to investigate EV biogenesis factors, marker expression, and localization across cell types in the skeletal muscle. We also aimed to investigate whether EV concentrations are altered by disuse atrophy.MethodsTo identify the potential markers of SkM-derived EVs, EVs were isolated from rat serum using density gradient ultracentrifugation, followed by fluorescence correlation spectroscopy measurements or qPCR. Single-cell RNA sequencing (scRNA-seq) data from rat SkM were analyzed to assess the EV biogenesis factor expression, and cellular localization of tetraspanins was investigated by immunohistochemistry. Finally, to assess the effects of mechanical unloading on EV expression in vivo, EV concentrations were measured in the serum by nanoparticle tracking analysis in both a rat and human model of disuse.ResultsIn this study, we show that the widely used markers of SkM-derived EVs, α-sarcoglycan and miR-1, are undetectable in serum EVs. We also found that EV biogenesis factors, including the tetraspanins CD63, CD9, and CD81, are expressed by a variety of cell types in SkM. SkM sections showed very low detection of CD63, CD9, and CD81 in myofibers and instead accumulation within the interstitial space. Furthermore, although there were no differences in serum EV concentrations following hindlimb suspension in rats, serum EV concentrations were elevated in human subjects after bed rest.ConclusionsOur findings provide insight into the distribution and localization of EVs in SkM and demonstrate the importance of methodological guidelines in SkM EV research.

Diagnostic performances of Schistosoma haematobium and Schistosoma mansoni recombinant proteins, peptides and chimeric proteins antibody based tests. Systematic scoping review.

Traditional diagnostic tests for schistosome infections are suboptimal, particularly when the parasite burden is low. In the present review we sought to identify recombinant proteins, peptides, and chimeric proteins with potential to be used as sensitive and specific diagnostic tools for schistosomiasis. The review was guided by PRISMA-ScR guidelines, Arksey and O'Malley's framework, and guidelines from the Joanna Briggs Institute. Five databases were searched: Cochrane library, PubMed, EMBASE, PsycInfo and CINAHL, alongside preprints. Identified literature were assessed by two reviewers for inclusion. A narrative summary was used to interpret the tabulated results. Diagnostic performances were reported as specificities, sensitivities, and AUC. The AUC for S. haematobium recombinant antigens ranged from 0.65 to 0.98, and 0.69 to 0.96 for urine IgG ELISA. S. mansoni recombinant antigens had sensitivities ranging from 65.3% to 100% and specificities ranging from 57.4% to 100%. Except for 4 peptides which had poor diagnostic performances, most peptides had sensitivities ranging from 67.71% to 96.15% and specificities ranging from 69.23% to 100%. S. mansoni chimeric protein was reported to have a sensitivity of 86.8% and a specificity of 94.2%. The tetraspanin CD63 antigen had the best diagnostic performance for S. haematobium. The tetraspanin CD63 antigen Serum IgG POC-ICTs had a sensitivity of 89% and a specificity of 100%. Peptide Smp_150390.1 (216-230) serum based IgG ELISA had the best diagnostic performance for S. mansoni with a sensitivity of 96.15% and a specificity of 100%. Peptides were reported to demonstrate good to excellent diagnostic performances. S. mansoni multi-peptide chimeric protein further improved the diagnostic accuracy of synthetic peptides. Together with the advantages associated with urine sampling technique, we recommend development of multi-peptide chimeric proteins urine based point of care tools.

Small extracellular vesicles have distinct CD81 and CD9 tetraspanin expression profiles in plasma from rheumatoid arthritis patients.

Extracellular vesicles (EVs) are implicated in the pathogenesis of rheumatoid arthritis (RA) but little is known about the composition of specific small EV (sEV) subpopulations. This study aimed to characterize the CD63, CD81 and CD9 tetraspanin profile in the membrane of single EVs in plasma from treatment naïve RA patients and assess potential discrepancies between methotrexate (MTX) responder groups. EVs isolated from plasma were characterized using transmission electron microscopy, and detection of surface markers (CD63, CD81 and CD9) on single EVs was performed on the ExoView platform. All RA patients (N = 8) were newly diagnosed, treatment naïve, females, ACPA positive and former smokers. The controls (N = 5) were matched for age and gender. After three months of MTX treatment, responders (N = 4) were defined as those with ΔDAS28 > 1.2 and DAS28 ≤ 3.2 post-treatment. The isolated EVs were 50-200nm in size. The RA patients had a higher proportion of both CD9 and CD81 single positive sEVs compared to healthy controls, while there was a decrease in CD81/CD9 double positive sEVs in patients. Stratification of RA patients into MTXresponders and non-responders revealed a distinctly higher proportion of CD81 single positive sEVs in the responder group. The proportion of CD81/CD9 double positive sEVs (anti-CD9 captured) was lower in the non-responders, but increased upon 3months of MTXtreatment. Our exploratory study revealed distinct tetraspanin profiles in RA patients suggesting their implication in RA pathophysiology and MTX treatment response.

Effects of MITF on marker protein expression of multivesicular bodies and miRNA omics of extracellular vesicles of mice melanocyte cell line

Extracellular vesicles (EVs) are heterogeneous membrane-bound complexes of cell-derived and nanosized structures originating from the endosomal system and subsequently released from the plasma membrane. EVs contribute significantly to intercellular communication and are involved in pigmentation processes that rely on tight communication between keratinocytes and melanocytes in the epidermis. Microphthalmia-associated transcription factor (MITF) induces melanogenesis and modulates the expression factors involved in melanosome biogenesis, maturation and dispersal in melanocytes. Here, we evaluated the effects of MITF on the fate of multivesicular bodies and the biogenesis of extracellular vesicles of melanocytes. It was found that MITF increased the expression of subunits of the endosomal sorting complex, required for transport (ESCRT), including VPS37, VPS36B, and tetraspanin CD81, which are key mediators of multivesicular body biogenesis. Over 110 miRNAs, including miR-211–5p, miR-335–5p, let-7g-5p and miR-28a-3p, were differentially expressed in melanocyte-derived EVs after overexpression of MITF in melanocytes. These miRNAs have been reported to be key regulators of plasma protein binding, changes in the cell membrane system and transferase activity. These results suggest that while enhancing melanogenesis, melanocytes may mediate intercellular communication with surrounding cells by serving as EV delivery vehicles.

Developmental trajectory of extracellular vesicle characteristics from the lungs of preterm infants.

Extracellular vesicles (EVs) are secreted lipid-enclosed particles that have emerged as potential biomarkers and therapeutic agents in lung disease, including bronchopulmonary dysplasia (BPD), a leading complication of preterm birth. Many unanswered questions remain about the content and cargo of EVs in premature infants and their role in lung development. To characterize EVs during human lung development, tracheal aspirates were collected from premature neonates between 22 and 35 wk gestational age and analyzed via nanoparticle tracking analysis, electron microscopy, and bead-based flow cytometry. EVs were detectable across late canalicular through saccular stages of lung development, demonstrating larger sizes earlier in gestation. EVs contained an abundance of the EV-enriched tetraspanins CD9, CD63, and CD81, as well as epithelial cell and immune cell markers. Increases in select surface proteins (CD24 and CD14) on EVs were associated with gestational age and with the risk of BPD. Finally, query of expression data obtained from epithelial cells in a single-cell atlas of murine lung development found that epithelial EV marker expression also changes with developmental time. Together, these data demonstrate an association between EV profile and lung development and provide a foundation for future functional classification of EVs, with the goal of determining their role in cell signaling during development and harnessing their potential as a new therapeutic target in BPD.

Antimicrobial Effects of Tetraspanin CD9 Peptide against Microbiota Causing Armpit Malodour.

Synthetic peptides, including tetraspanin CD9 peptides, are increasingly coming into focus as new treatment strategies against various organisms, including bacteria, that cause underarm odour. The use of deodorants and antiperspirants is associated with side effects. Therefore, it is critical to find an alternative therapeutic approach to combat underarm odour. The aim of this study is to investigate the antibacterial effect of tetraspanin CD9 peptides against the skin microbiota that cause malodour in the underarms. The antimicrobial activity of CD9 peptides against Micrococcus luteus (M. luteus), Bacillus subtilis (B. subtilis), Staphylococcus epidermidis (S. epidermidis), and Corynebacterium xerosis (C. xerosis) was investigated by the disc diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution assays using CD9 peptide concentrations ranging from 1 mg/mL to 0.0078 mg/mL. In addition, the anti-biofilm activity of the CD9 peptides was determined. The CD9 peptides showed different antibacterial activity with an inhibition zone of 7.67, 9.67, 7.00, and 6.00 mm for S. epidermidis, M. luteus, C. xerosis, and B. subtilis, respectively. All bacteria had the same MBC value of 1 mg/mL. A high MIC of CD9 peptides was observed for S. epidermidis and M. luteus at 0.5 mg/mL. The MIC values of B. subtilis and C. xerosis were 0.125 mg/mL and 0.25 mg/mL, respectively. CD9 peptides significantly inhibited biofilm development of S. epidermidis, B. subtilis, and C. xerosis isolates. The CD9 tetraspanin peptide has excellent antibacterial activity against bacteria that cause underarm odour. Therefore, the CD9 tetraspanin peptide is a promising alternative to deodorants and antiperspirants to combat commensal bacteria of the skin that cause underarm odour.

A specific technique of immunolabelling of urinary small extracellular vesicle biomarkers for the diagnostic of renal cancer.

Small extracellular vesicles (EVs) are characterized by the membrane expression of CD63, CD81 and CD9 tetraspanins. Their size is inferior to 200 nm. They share the same characteristics as the native cells and are found in human fluids. Specific membrane protein biomarkers expressed on small EV are useful for the diagnosis of tumoural pathologies. Clear cell renal cell carcinoma (CCRCC) is diagnosed by imaging examinations and/or tissue biopsy. Carbonic anhydrase IX (CAIX) is a powerful biomarker of CCRCC. The detection of CAIX on small EV from the urine of patients could constitute a liquid biopsy for CCRCC. We have set up a specific protocol for the preparation, the immunostaining characterization and the transmission electron microscopy observation of small EVs isolated from the urine of CCRCC patients. The background labelling was significantly reduced. We successfully detected biomarkers on urinary small EVs from CCRCC patients. This technique could be extended with antibodies directed against other EV biomarkers for the detection and the monitoring of cancer diseases.

Mycobacterium abscessus alkyl hydroperoxide reductase C promotes cell invasion by binding to tetraspanin CD81

Mycobacterium abscessus (Mab) is an increasingly recognized pulmonary pathogen. How Mab is internalized by macrophages and establishes infection remains unknown. Here, we show that Mab uptake is significantly reduced in macrophages pre-incubated with neutralizing anti-CD81 antibodies or in cells in which the large extracellular loop (LEL) of CD81 has been deleted. Saturation of Mab with either soluble GST-CD81-LEL or CD81-LEL-derived peptides also diminished internalization of the bacilli. The mycobacterial alkyl hydroperoxide reductase C (AhpC) was unveiled as a major interactant of CD81-LEL. Pre-exposure of macrophages with soluble AhpC inhibited mycobacterial uptake whereas overexpression of AhpC in Mab enhanced its internalization. Importantly, pre-incubation of macrophages with anti-CD81-LEL antibodies inhibited phagocytosis of AhpC-coated beads, indicating that AhpC is a direct interactant of CD81-LEL. Conditional depletion of AhpC in Mab correlated with decreased internalization of Mab. These compelling data unravel a previously unexplored role for CD81/AhpC to promote uptake of pathogenic mycobacteria by host cells.

A peptide binding to the tetraspanin CD9 reduces cancer metastasis

As an organizer of multi-molecular membrane complexes, the tetraspanin CD9 has been implicated in a number of biological processes, including cancer metastasis, and is a candidate therapeutic target. Here, we evaluated the suppressive effects of an eight-mer CD9-binding peptide (CD9-BP) on cancer cell metastasis and its mechanisms of action. CD9-BP impaired CD9-related functions by adversely affecting the formation of tetraspanin webs—networks composed of CD9 and its partner proteins. The anti-cancer metastasis effect of CD9-BP was evidenced by the in vitro inhibition of cancer cell migration and invasion as well as exosome secretion and uptake, which are essential processes during metastasis. Finally, using a mouse model, we showed that CD9-BP reduced lung metastasis in vivo. These findings provide insight into the mechanism by which CD9-BP inhibits CD9-dependent functions and highlight its potential application as an alternative therapeutic nano-biomaterial for metastatic cancers.

Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.