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Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal surface protein composition of heterogeneous EV populations in culture supernatants and in biofluids. Tetraspanins CD9, CD63, and CD81 are common and abundant EV markers. However, their relative enrichment (profile) on EVs of specific cellular origins is not fully elucidated. We introduce LuminEV, a novel version of the Luminex assay for the multiplexed analysis of EV surface proteins. Optimized LuminEV reagents enable direct, specific, and sensitive measurements of EV markers in biofluids and in culture supernatants, bypassing EV isolation step. LuminEV assay for CD9, CD63, and CD81 was validated by comparing simplex and multiplex measurements, establishing linearity, spike-in recovery, inter- and intra-assay precision, and reproducibility between operators. LuminEV measurements of CD9, CD63, and CD81 in conditioned media from 15 cell lines revealed strong variations between cell types and showed high sensitivity, which enabled EV detection without prior concentration. Using tetraspanin levels as a readout, we noted suppression and induction of EV release from the cultured cells by GW6869 and monensin. Measurement of EV CD9, CD63, and CD81 in blood plasma from 70 disease-free donors showed respective abundance of 72, 16, and 12%. CD63 displayed weak, albeit significant, negative correlation with age and was slightly lower in female samples. The assay was then used to detect cell type-specific EV surface markers, including CD235a (erythrocytes), GAP43 (neurons), and CD68 (macrophages), and to detect differences in tetraspanin profiles between healthy and diseased donors. In summary, LuminEV offers robust and sensitive approach for multiplexed assessment of EV surface proteins, to facilitate the research into EV biology, biomarker, and therapeutic applications., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

A new, sensitive, and cost-effective lab-on-paper-based immunosensor was designed based on electrochemical impedance spectroscopy (EIS) for the detection of exosomes. EIS was selected as the determination method since there was a surface blockage in electron transfer by binding the exosomes to the transducer. Briefly, the carbon working electrode (WE) on the paper electrode (PE) was modified with gold particles (AuPs@PE) and then conjugated with anti-CD9 (Anti-CD9/AuPs@PE) for the detection of exosomes. Variables involved in the biosensor design were optimized with the univariate mode. The developed method presents the limit of detection of  8.7 × 10 2 exosomes mL -1 , which is lower than that of many other available methods under the best conditions. The biosensor was also tested with urine samples from cancer patients with high recoveries. Due to this  a unique, low-cost, biodegradable technology is presented that can directly measure exosomes without labeling them for early cancer or metastasis detection., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na 7 PMo 11 O 39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na 7 PMo 11 O 39 NPs. The Au/Na 7 PMo 11 O 39 -CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na 7 PMo 11 O 39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 10 7 -2.81 × 10 8 particles/mL, with a limit of detection (LOD) of 5.82 × 10 7 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Objective: Extracellular vesicles (EV) 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, EV present themselves as sentinels of function and damage, providing a non-invasive approach. However, the analysis of EV in urine requires prior isolation, which slows down and hinders transition into 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 EV and proteins involved in renal function., Materials and Methods: The ExoView® technology enables the quantification and phenotyping of EV present in urine and the quantification of their membrane and internal proteins. We have applied this technology to the quantification of urinary EV and their proteins with renal tubular expression, amnionless (AMN) and secreted frizzled-related protein 1 (SFRP1), using only 5 μl of urine. Tubular expression was confirmed by immunohistochemistry., Results: The mean size of the EV analysed was 59 ± 16 nm for those captured by tetraspanin CD63, 61 ± 16 nm for those captured by tetraspanin CD81, and 59 ± 10 for tetraspanin CD9, with CD63 being the majority EV 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)., Conclusions: This work demonstrates the applicability and advantages of the ExoView® technique for the direct analysis of urine EV and their protein content in relation to the renal tubule. The use of minimum volumes, 5 μl, and the total analysis time not exceeding three hours facilitate the transition of EV into daily clinical practice as sources of diagnostic information., (Copyright © 2023 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.)

Exosomes, as an emerging biomarker, have exhibited remarkable promise in early cancer diagnosis. Here, a highly sensitive, selective, and automatic electrochemiluminescence (ECL) method for the detection of cancerous exosomes was developed. Specific aptamer-(EK) 4 peptide-tagged magnetic beads (MBs-(EK) 4 -aptamer) were designed as a magnetic capture probe in which the (EK) 4 peptide was used to reduce the steric binding hindrance of cancerous exosomes with a specific aptamer. One new universal ECL signal nanoprobe (CD9 Ab-PEG@SiO 2 ϵRu(bpy) 3 2+ ) was designed and synthesized by using microporous SiO 2 nanoparticles as the carrier for loading ECL reagent Ru(bpy) 3 2+ , polyethylene glycol (PEG) layer, and anticluster of differentiation 9 antibody (CD9 Ab). A "sandwich" biocomplex was formed on the surface of the magnetic capture probe after mixing the capture probe, target exosomes, and ECL signal nanoprobe, and then it was introduced into an automated ECL analyzer for rapid and automatic ECL measurement. It was found that the designed signal nanoprobe shows a 270-fold improvement in the signal-to-noise ratio than that of the ruthenium complex-labeled CD9 antibody signal probe. The relative ECL intensity was proportional to MCF-7 exosomes as a model in the range of 10 2 to 10 4 particle/μL, with a detection limit of 11 particle/μL. Furthermore, the ECL method was employed to discriminate cancerous exosomes based on fingerprint responses using the designed multiple magnetic capture probes and the universal ECL signal nanoprobe. This work demonstrates that the utilization of a designed automated ECL tactic using the MBs-(EK) 4 -aptamer capture probe and the CD9 Ab-PEG@SiO 2 ϵRu(bpy) 3 2+ signal nanoprobe will provide a unique and robust method for the detection and discrimination of cancerous exosomes.

Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs' affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 10 4 particles·mL -1 to 6.1 × 10 7 particles·mL -1 , and a low detection limit (0.6-1.8) × 10 4 particles·mL -1 . A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.

Background/aim: Exosomes secreted by various cells in the tumour microenvironment have been reported to be mediators of intercellular communication that play an important role in cancer progression. In this study, we aimed to investigate the effects of exosomes derived from cancer-associated fibroblasts (CAFs) on the proliferation of malignant melanoma (MM) cells and evaluated their clinicopathological significance., Materials and Methods: Three malignant melanoma cell lines, A375, MMAc, and COLO679, and three CAFs established from malignant melanomas at stages 1a, 2b, and 3b, were used. The expression of CD9, CD63, and CD81 in CAF-derived exosomes was examined using western blotting. The effect of exosomes on the proliferative potential of cancer cells was analysed using cell counting and MTT assays. The expression of CD9, CD63, and CD81 was also immunohistochemically analysed in 90 malignant melanoma specimens., Results: CAF-derived exosomes were positive for CD9 and CD63 and remarkably inhibited the proliferative capacity of A375 and MMAc cells. The five-year disease-free survival was significantly better in patients with CAF-derived CD9-positive exosomes than in CD9-negative patients., Conclusion: CAF-derived exosomes, especially CD9-positive exosomes, have an inhibitory effect on the proliferation of malignant melanoma cells. These findings suggest that CD9 expression in CAFs is a promising prognostic marker for patients with malignant melanoma., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Primary aldosteronism (PA) is caused by autonomous overproduction of aldosterone, which induces organ damage directly via activation of the mineralocorticoid receptor (MR); however, no specific or sensitive biomarkers are able to reflect MR activity. Recently, it is found that urinary extracellular vesicles (uEVs) are secreted by multiple cell types in the kidney and are an enriched source of kidney-specific proteins. Here, we evaluate sodium transporters in uEVs as candidates of biomarkers of MR activity in the clinical setting. Sixteen patients were examined to determine their plasma aldosterone concentration (PAC) and renin activity, and their morning urine was collected. The protein levels of two sodium transporters in uEVs, γ-epithelial sodium channel (γENaC) and thiazide-sensitive sodium chloride cotransporter (NCC), were quantified by Western blot analysis, and their clinical correlation with PAC was determined. Consequently, we found PAC was significantly correlated with the γENaC protein level adjusted by the CD9 protein level in uEVs (correlation coefficient = 0.71). PAC was also correlated with the NCC protein level adjusted by the CD9 protein level in uEVs (correlation coefficient = 0.61). In two PA patients, treatment with an MR antagonist or adrenalectomy reduced γENaC/CD9 in uEVs. In conclusion, γENaC/CD9 in uEVs is a valuable biomarker of MR activity in PA patients and may be a useful biomarker for other MR-associated diseases.

We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of the sensor surface in a range -16.0 to -32.8 mV. Thereafter, we examine the sensitivity of the sensor surface across this range of zeta potential to determine the optimal functionalization scheme. The limit of detection (LOD) varied by 2 orders of magnitude across this range, reaching a value of 4.9 × 10 6 particles/mL for the best performing surface for CD9. We then used the optimized surface to profile CD9, EGFR, and PD-L1 surface proteins of sEVs derived from non-small cell lung cancer (NSCLC) cell-line H1975, before and after treatment with EGFR tyrosine kinase inhibitors, as well as sEVs derived from pleural effusion fluid of NSCLC adenocarcinoma patients. Our results show the feasibility to monitor CD9, EGFR, and PD-L1 expression on the sEV surface, illustrating a good prospect of the method for clinical application.

In this work, we report on two novel monoclonal antibodies, specific for porcine CD9. CD9 is a tetraspanin that is expressed on a wide variety of cells. We phenotyped porcine immune cell subsets and found that CD9 was expressed on all monocytes as well as a subset of B cells. CD9 was variably expressed on T cells, with CD4 T cells containing the highest frequency of CD9 + cells. CD9 expression positively correlated with the frequency of central memory CD4 T cells in ex vivo PBMC. Therefore, we proceeded to explore CD9 as a marker of T cell function. Here we observed that CD9 was expressed on the vast majority of long-lived influenza A virus-specific effector cells that retained the capacity for cytokine production in response to in vitro recall antigen. Therefore, the new antibodies enable the detection of a cell surface molecule with functional relevance to T cells. Considering the importance of CD9 in membrane remodelling across many cell types, they will also benefit the wider field of swine biomedical research., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo , CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Although small extracellular vesicles (sEVs) carry DNA, miRNA, and proteins, and they play an important role in long-distance intercellular communication, their generation and circulation mechanisms are unclear. sEVs can be used as biomarkers for the early diagnosis of diseases (e.g., cancer, Alzheimer's disease, melanoma, and cardiovascular diseases) and as drug delivery carriers to the target tissues. Hence, sEVs are attracting considerable attention from scientists and medical professionals. In the present study, we investigated four different commercially available cationic particles (two silica particles modified with diethylaminopropyl or trimethylaminopropyl groups, and two agarose particles modified with diethylaminopropyl or trimethylaminopropyl groups) for the purification of sEVs obtained from a cell culture medium. All the cationic particles captured the sEVs well. The NaCl concentrations required for elution of the captured sEVs differed for the different cationic particles. sEVs were most efficiently captured by silica particles modified with diethylaminopropyl groups, and they were eluted from these particles using 200 mM NaCl as the elution solution. Because the developed method can be used to easily purify sEVs obtained from a culture medium, it is expected to facilitate the functional analysis of sEVs, as well as early diagnosis and treatment of diseases using sEVs.

CD9 is a transmembrane glycoprotein belonging to the tetraspanin family. CD9 expression has been reported to be associated with cellular signaling, cell adhesion, cell migration, and tumor related processes. The aim of this study was to examine the immunohistochemical expression of CD9 in vascular senescence and atherosclerosis. One hundred and twenty samples of normal young arteries (obtained from individuals aged 0-60 years), 40 samples of normal old arteries (obtained from individuals aged 61-80 years), and 67 samples of atherosclerotic arteries were obtained from surgically resected specimens. Tissue microarray blocks were prepared for immunohistochemical staining. Immunohistochemical staining detected CD9 expression in 10.8% (13 of 120 samples) of normal young arteries and 30.0% (12 of 40 samples) of normal old arteries. CD9 expression was absent or mildly present in the smooth muscle cells and endothelial cells of normal arteries. Normal old arteries showed significantly higher expression of CD9 than normal young arteries (P<0.01). Atherosclerotic arteries showed moderate or strong CD9 expression (65 of 67 samples, 97.0%), which was observed in the smooth muscle cells, endothelial cells, macrophages, and atheromatous plaques. CD9 was significantly expressed in the atherosclerotic arteries compared to normal young and old arteries (P<0.01). The results suggest that CD9 expression may play an important role in the vascular senescence and pathogenesis of atherosclerosis.

Extracellular vesicles (EVs), naturally occurring nanosized vesicles secreted from cells, are essential for intercellular communication. They carry unique biomolecules on the surface or interior that are of great interest as biomarkers for various pathological conditions such as cancer. In this work, we use high-resolution atomic force microscopy (AFM) and spectroscopy (AFS) techniques to demonstrate differences between EVs derived from colon cancer cells and colon epithelial cells at the single-vesicle level. We observe that EV populations are significantly increased in the cancer cell media compared to the normal cell EVs. We show that both EVs display an EV marker, CD9, while EVs derived from the cancer cells are slightly higher in density. Hyaluronan (HA) is a nonsulfated glycosaminoglycan linked to malignant tumor growth according to recent reports. Interestingly, at the single-vesicle level, colon cancer EVs exhibit significantly increased HA surface densities compared to the normal EVs. Spectroscopic measurements such as Fourier transform infrared (FT-IR), circular dichroism (CD), and Raman spectroscopy unequivocally support the AFM and AFS measurements. To our knowledge, it represents the first report of detecting HA-coated EVs as a potential colon cancer biomarker. Taken together, this sensitive approach will be useful in identifying biomarkers in the early stages of detection and evaluation of cancer.

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 10 8 /ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 10 8 /ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 10 8 /ml) was higher compared to that of blastocysts (5.68 × 10 8 /ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals., (© 2020 Blackwell Verlag GmbH.)

Seminal extracellular vesicles (EVs) include exosomes (ø 40-120 nm) and microvesicles (MVs, ø 120-1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs (P < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher (P < 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.

Flow cytometry provides robust, multi-parametric and quantitative information on single cells which also exhibits enormous potential as a tool for small particle characterisation. Small extracellular vesicle (sEV) detection by flow cytometry remains compromised due to the high prevalence of swarm detection, which is defined by the simultaneous illumination of more than one sEV, recorded as a single event. Detection of sEVs by imaging flow cytometry presents a major advantage by having the ability to resolve single particles from swarm detection based on the image features recorded for each event. In this study, we provide a simplified protocol that facilitates the removal of both swarm events and aggregated particles to improve the accuracy of sEV analysis. Our results indicate that imaging flow cytometry should be at the forefront as a robust and sensitive technique for sEV characterisation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To determine if prostate-derived extracellular vesicles (EVs) present in patient plasma samples are of exocytotic origin (exosomes) or released by the cell membrane (microparticles/microvesicles). Both malignant and normal prostate cells release two types of EVs into the circulation, exosomes, and microparticles/microvesicles which differ in size, origin, and mode of release. Determining what proportion of prostate-derived EVs are of exosomal versus microparticle/microvesicle EV subtype is of potential diagnostic significance., Materials and Methods: Multi-parametric analytical platforms such as nanoscale flow cytometry (nFC) were used to analyze prostate derived extracellular vesicles. Plasmas from prostate cancer (PCa) patient plasmas representing benign prostatic hyperplasia (BPH), low grade prostate cancer (Gleason Score 3 + 3) and high grade prostate cancer (Gleason Score ≥4 + 4) were analyzed for various exosome markers (CD9, CD63, CD81) and a prostate specific tissue marker (prostate specific membrane antigen/PSMA)., Results: By using nanoscale flow cytometry, we determine that prostate derived EVs are primarily of cell membrane origin, microparticles/microvesicles, and not all PSMA expressing EVs co-express exosomal markers such as CD9, CD63, and CD81. CD9 was the most abundant exosomal marker on prostate derived EVs (12-19%). There was no trend observed in terms of more PSMA + CD9 or PSMA + CD63 co-expressing EVs versus increasing grade of prostate cancer., Conclusion: The majority of prostate derived EVs present in plasmas are from the cell membrane as evidenced by their size and most importantly, lack of co-expression of exosomal markers such as CD9/CD63/CD81. In fact, CD81 was not present on any prostate derived EVs in patient plasmas whereas CD9 was present on a minority of prostate derived EVs. The addition of an exosomal marker for detection of prostate-derived EVs does not provide greater clarity in distinguishing EVs released by the prostate., (© 2019 Wiley Periodicals, Inc.)

Mammalian fertilization involves membrane events-adhesion, fusion, sperm engulfment, membrane block to polyspermy-whose causes remain largely unknown. Recently, specific oscillations of the sperm in contact with the egg were shown to be necessary for fusion. Using a microfluidic chip to impose the venue for the encounter of two gametes allowed real-time observation of the membrane remodelling occurring at the sperm/egg interface. The spatiotemporal mapping of egg CD9 revealed that this protein concentrates at the egg/sperm interface as a result of sperm oscillations, until a CD9-rich platform is nucleated on which fusion immediately takes place. Within 2-5 min after fusion, most of the CD9 leaves the egg for the external aqueous medium. Then an egg membrane wave engulfs the sperm head in ~25 min. These results show that sperm oscillations initiate the CD9 recruitment that causes gamete fusion after which CD9 and associated proteins leave the membrane in a process likely to contribute to block polyspermy. They highlight that the gamete fusion story in mammals is an unexpected interplay between mechanical constraints and proteins.

To facilitate intercellular communication, cells release nano-sized, extracellular vesicles (EVs) to transfer biological cargo to both local and distant sites. EVs are enriched in tetraspanins, two of which (CD9 and CD151) have altered expression patterns in many solid tumours, including prostate cancer, as they advance toward metastasis. We aimed to determine whether EVs from prostate cells with altered CD9 and CD151 expression could influence cellular behaviour and increase the metastatic capabilities of non-tumourigenic prostate cells. EVs were isolated by ultrafiltration and characterised for their tetraspanin expression and size distribution. iTRAQ was used to identify differences between RWPE1 and tetraspanin-modified RWPE1 EV proteomes, showing an enrichment in protein degradation pathways. Addition of EVs from RWPE1 cells with reduced CD9 or increased CD151 abundance resulted in increased invasion of RWPE1 cells, and increased migration in the case of high CD151 abundance. We have been able to show that alteration of CD9 and CD151 on prostate cells alters the proteome of their resultant EVs, and that these EVs can enhance the migratory and invasive capabilities of a non-tumourigenic prostate cellular population. This work suggests that cellular tetraspanin levels can alter EVs, potentially acting as a driver of metastasis in prostate cancer.

Background & Objectives: CD9 and CD146 are important adhesion molecules that play a role in the implantation of an embryo. This study was undertaken to correlate the expression of these markers in fertile and infertile women's endometrial stromal cells., Methods: Human endometrial stromal cell culture from endometrial biopsies of fertile (n=50) and infertile females (n=50) was performed and primary cell lines were established. Expression of CD9 and CD146 was studied for all the 100 cell lines with the help of flow cytometry. Gene expression of CD9 and CD146 was performed by real-time polymerase chain reaction., Results: There was a significant difference in endometrial stromal cells of fertile and infertile females. Flow cytometric results revealed significantly lower expression of CD9 (P=0.0126) and CD146 (P=0.0006) in the infertile endometrial stromal cells as compared to fertile endometrial stromal cells. These results were comparable with real-time data., Interpretation & Conclusions: This study showed that endometrial stromal cells from infertile females had lower expression of adhesion molecules, CD9 and CD146. Our findings suggest that CD9 and CD146 may have a role in infertility. Infertile female's endometrial stromal cells have decreased expression of CD9 and CD146 which can be the cause of infertility related to implantation failure., Competing Interests: None

Obesity is characterized by an accumulation of macrophages in adipose, some of which form distinct crown-like structures (CLS) around fat cells. While multiple discrete adipose tissue macrophage (ATM) subsets are thought to exist, their respective effects on adipose tissue, and the transcriptional mechanisms that underlie the functional differences between ATM subsets, are not well understood. We report that obese fat tissue of mice and humans contain multiple distinct populations of ATMs with unique tissue distributions, transcriptomes, chromatin landscapes, and functions. Mouse Ly6c ATMs reside outside of CLS and are adipogenic, while CD9 ATMs reside within CLS, are lipid-laden, and are proinflammatory. Adoptive transfer of Ly6c ATMs into lean mice activates gene programs typical of normal adipocyte physiology. By contrast, adoptive transfer of CD9 ATMs drives gene expression that is characteristic of obesity. Importantly, human adipose tissue contains similar ATM populations, including lipid-laden CD9 ATMs that increase with body mass. These results provide a higher resolution of the cellular and functional heterogeneity within ATMs and provide a framework within which to develop new immune-directed therapies for the treatment of obesity and related sequela., Competing Interests: The authors declare no conflict of interest.

Proteins CD9 and CD81 are members of the tetraspanin superfamily and were detected in mammalian sperm, where they are suspected to form an active tetraspanin web and to participate in sperm⁻egg membrane fusion. The importance of these two proteins during the early stages of fertilization is supported by the complete sterility of CD9/CD81 double null female mice. In this study, the putative mechanism of CD9/CD81 involvement in tetraspanin web formation in sperm and its activity prior to fertilization was addressed. Confocal microscopy and colocalization assay was used to determine a mutual CD9/CD81 localization visualised in detail by super-resolution microscopy, and their interaction was address by co-immunoprecipitation. The species-specific traits in CD9 and CD81 distribution during sperm maturation were compared between mice and humans. A mutual position of CD9/CD81 is shown in human spermatozoa in the acrosomal cap, however in mice, CD9 and CD81 occupy a distinct area. During the acrosome reaction in human sperm, only CD9 is relocated, compared to the relocation of both proteins in mice. The structural modelling of CD9 and CD81 homologous and possibly heterologous network formation was used to propose their lateral Cis as well as Trans interactions within the sperm membrane and during sperm⁻egg membrane fusion.

Extracellular vesicles (EVs) have stimulated considerable scientific and clinical interest, yet protein profiling and sizing of individual EVs remains challenging due to their small particle size, low abundance of proteins, and overall heterogeneity. Building upon a laboratory-built high-sensitivity flow cytometer (HSFCM), we report here a rapid approach for quantitative multiparameter analysis of single EVs down to 40 nm with an analysis rate up to 10 000 particles per minute. Statistically robust particle size distribution was acquired in minutes with a resolution and profile well matched with those of cryo-TEM measurements. Subpopulations of EVs expressing CD9, CD63, and/or CD81 were quantified upon immunofluorescent staining. When HSFCM was used to analyze blood samples, a significantly elevated level of CD147-positive EVs was identified in colorectal cancer patients compared to healthy controls (P < 0.001). HSFCM provides a sensitive and rapid platform for surface protein profiling and sizing of individual EVs, which could greatly aid the understanding of EV-mediated intercellular communication and the development of advanced diagnostic and therapeutic strategies.

Objective: We undertook a single-center retrospective study to determine the relationship between CD9 and acute lymphoblastic leukemia (ALL)., Materials and Methods: In total, 112 newly diagnosed patients in our center were enrolled in the study. Their clinical information was collected and the patients werefollowed over the course of the study. Flow cytometry was used to detect the expression of CD9., Results: CD9 expression was more common in B cell acute lymphoblastic leukemia (B-ALL) and patients > 40 years old. CD9-positive patients exhibited a higher BCR-ABL fusion gene positive rate and higher neutrophil counts than CD9 negative patients (P= 0.004 and P= 0.004, respectively). Response to induction chemotherapy was not dependent on CD9 expression. CD9-positive patients had a lower 2-year overall survival rate than CD9-negative patients., Conclusion: CD9 expression predicts some clinical characteristics and indicates an unfavorable prognosis in ALL patients.

Objectives: Exosomes are important mediators in intercellular communications and play a role in cancer progression and metastasis. Exosomal membranes are enriched in endosome-specific tetraspanins (CD9 and CD63). Here, we explored the expression of CD63 and CD9 utilizing immunohistochemistry in malignant and nonmalignant cells in 29 resected pancreatic specimens (RPSs) of mixed racial background., Methods: The pathologic tissues (PTs) and adjacent normal tissues (ANTs) in each RPS were stained for CD63 and CD9. Two pathologists independently scored the expression of CD63 and CD9. Staining intensity was graded from 1 to 3. Staining percentage was estimated in 10% increments. An average Quick score (Q score) (intensity × percentage of staining) was calculated. Unpaired t test was used for statistical analysis., Results: The mean multiplicative Q score for CD63 and CD9 expression is higher in PTs (209 and 72) compared with ANTs (154 and 24) (P = 0.0041, P = 0.0018), respectively. The Mean Q score for CD63 and CD9 expression is higher in the malignant PTs (231 and 85) compared with ANTs (129 and 25) (P < 0.0001 and P < 0.0124)., Conclusions: Exosomal markers (CD63 and CD9) expression assessment using immunohistochemistry is feasible in RPS. The expression of CD63 and CD9 is higher in PTs and malignant PTs compared with their ANTs.

Aims: CD9, a tetraspanin transmembrane protein, modulates cell motility, migration, and proliferation. The aim of this study was to investigate the prognostic significance of CD9 expression in patients with invasive breast carcinoma (IBC)., Methods and Results: CD9 expression was evaluated in tissue microarrays of 1349 IBC samples via immunohistochemistry. CD9 expression in tumour cells (T-CD9 expression) and CD9 expression in stromal immune cells (S-CD9 expression) were analysed separately. T-CD9 expression was observed in 732 (54.3%) cases, and was associated with lymph node metastasis, histological type, lymphovascular invasion, high histological grade, HER2 positivity, a high Ki67 labelling index, and distant metastasis. S-CD9 expression was observed in 833 (61.7%) cases, and was associated with large tumour size, histological type, high histological grade, negative hormone receptors, HER2 positivity, a high Ki67 labelling index, and tumour-infiltrating lymphocytes. Patients with T-CD9 expression had shorter disease-free survival (DFS) than those without T-CD9 expression in the univariate and multivariate analyses. However, S-CD9 expression correlated significantly with a favourable DFS in the univariate and multivariate analyses. In the subgroup analysis, T-CD9 expression and S-CD9 expression were independent markers for DFS in luminal A and luminal B (HER2-negative) subgroups, respectively., Conclusions: T-CD9 expression could be a biomarker for poor prognosis in luminal A IBC, whereas S-CD9 expression could be a marker of good prognosis in luminal B (HER2-negative) IBC. Therefore, tumour compartment-specific analyses considering molecular subtypes are necessary to study the prognostic significance of CD9 expression in IBC., (© 2017 John Wiley & Sons Ltd.)

Extracellular vesicles (EVs), including exosomes, are circulating nanoscale particles heavily implicated in cell signaling and can be isolated in vast numbers from human biofluids. Study of their molecular profiling and materials properties is currently underway for purposes of describing a variety of biological functions and diseases. However, the large, and as yet largely unquantified, variety of EV subpopulations differing in composition, size, and likely function necessitates characterization schemes capable of measuring single vesicles. Here we describe the first application of multispectral optical tweezers (MS-OTs) to single vesicles for molecular fingerprinting of EV subpopulations. This versatile imaging platform allows for sensitive measurement of Raman chemical composition (e.g., variation in protein, lipid, cholesterol, nucleic acids), coupled with discrimination by fluorescence markers. For exosomes isolated by ultracentrifugation, we use MS-OTs to interrogate the CD9-positive subpopulations via antibody fluorescence labeling and Raman spectra measurement. We report that the CD9-positive exosome subset exhibits reduced component concentration per vesicle and reduced chemical heterogeneity compared to the total purified EV population. We observed that specific vesicle subpopulations are present across exosomes isolated from cell culture supernatant of several clonal varieties of mesenchymal stromal cells and also from plasma and ascites isolated from human ovarian cancer patients.

Extracellular vesicles (EVs) are membrane-bound nanovesicles delivered by different cellular lineages under physiological and pathological conditions. Although these vesicles have shown relevance as biomarkers for a number of diseases, their isolation and detection still has several technical drawbacks, mainly related with problems of sensitivity and time-consumed. Here, we reported a rapid and multiple-targeted lateral flow immunoassay (LFIA) system for the detection of EVs isolated from human plasma. A range of different labels (colloidal gold, carbon black and magnetic nanoparticles) was compared as detection probe in LFIA, being gold nanoparticles that showed better results. Using this platform, we demonstrated that improvements may be carried out by incorporating additional capture lines with different antibodies. The device exhibited a limit of detection (LOD) of 3.4×10 6 EVs/µL when anti-CD81 and anti-CD9 were selected as capture antibodies in a multiple-targeted format, and anti-CD63 labeled with gold nanoparticles was used as detection probe. This LFIA, coupled to EVs isolation kits, could become a rapid and useful tool for the point-of-care detection of EVs, with a total analysis time of two hours., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs), namely, exosomes and microvesicles, are important mediators of intercellular communication pathways. Since EVs can be detected in a variety of biofluids and contain a specific set of biomarkers which are reminiscent of their parental cells, they show great promise in clinical diagnostics as EV analysis can be performed in minimally invasive liquid biopsies. However, reliable, fast and cost-effective methods for their determination are still needed, especially if decentralized analysis is intended. In this study, we developed an electrochemical biosensor which works with 1.5 μL sample volume and can detect as low as 200 exosomes per microliter, with a linear range spanning almost 4 orders of magnitude. The sensor is specific and readily differentiates exosomes from microvesicles in samples containing 1000-fold excess of the latter. Capability of detecting exosomes in real samples (diluted serum) was shown. This was achieved by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for detection of captured exosomes. Signal amplification is presumably obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. Detection is performed based on electrochemical reduction of 3,3',5,5'-tetramethyl benzidine (TMB) after addition of horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for EV determination.

Myxomas are the most common non-invasive but life-threatening cardiac neoplasms due to obstruction of heart chambers and risk of embolism in a manner resembling thromboembolism as well. They can occasionally disseminate via their detached fragments into the bloodstream to seed and grow as secondary still benign tumors. In this study we evaluated morphological and clinical aspects of 14 ancient, degenerated left or right-sided cardiac atrial myxomas with expression of CD9 and CD63, which are found to contribute to platelet activation, aggregation and, as a result, intratumoral thrombosis or fragmentation. The appearance of tumors varied from sessile to polypoid revealing that a higher rate of endocardial thrombosis was associated with sessile compared to polypoid myxomas and left-sided tumors compared to right-sided ones in our study. In the general aspect of ancient calcifications, amorphous calcification with intra-tumor thrombosis was noted more frequently in sessile tumors, while well-formed osseous metaplasia was usually a feature of polypoid tumors. In our material osseous metaplasia did not coexist with massive thrombosis and was found in polypoid, pedunculated myxomas. Most importantly, CD9 overexpression was recorded in every studied myxoma and CD63 gave a weak reaction in myxoma cells.

ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC., (© The Author(s) 2015.)

Objective: Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer deaths worldwide. CD9 has been reported to play a critical role in cell motility, growth and metastasis of multiple cancers. The present study investigated the clinicopathological features of CD9, and its biological characteristics in ESCC., Methods: Fifteen normal esophageal tissue specimens, fifty-three ESCC adjacent tissues and one hundred and four ESCC tissues were included in this study. Using immunohistochemistry (IHC), the expression levels of CD9 were evaluated among different samples. And its clinicopathological parameters and its prognostic factors were analyzed. Western blotting was used to measure CD9 expression and colony formation was performed to determine the effect of CD9 on cell growth in ESCC TE-1 cells., Results: Compared with normal esophageal tissues and tumor adjacent tissues, CD9 expression level is significantly higher in ESCC tissues. CD9 expression correlated with tumor stage (P=0.022) and lymph node metastasis (P=0.019) in ESCC patients. Furthermore, the small interfering RNA-mediated silencing of CD9 expression in TE-1 cells resulted in increased proliferation as evidenced by increased colony number and colony size., Conclusion: CD9 expression is upregulated in ESCC tissues and its expression is correlated with tumor stage and lymph node metastasis in ESCC patients. CD9 suppresses the proliferation of TE-1 cells. CD9 may present a potential in tumor progression in ESCC.

STING (stimulator of IFN genes) activates the IFN-dependent innate immune response to infection on sensing the presence of DNA in cytosol. The quantity of STING accumulating in cultured cells varies; it is relatively high in some cell lines [e.g., HEp-2, human embryonic lung fibroblasts (HEL), and HeLa] and low in others (e.g., Vero cells). In a preceding publication we reported that STING was stable in four cell lines infected with herpes simplex virus 1 and that it was actively stabilized in at least two cell lines derived from human cancers. In this report we show that STING is exported from HEp-2 cells to Vero cells along with virions, viral mRNAs, microRNAs, and the exosome marker protein CD9. The virions and exosomes copurified. The quantity of STING and CD9 exported from one cell line to another was inoculum-size-dependent and reflected the levels of STING and CD9 accumulating in the cells in which the virus inoculum was made. The export of STING, an innate immune sensor, and of viral mRNAs whose major role may be in silencing viral genes in latently infected neurons, suggests that the virus has evolved mechanisms that curtail rather than foster the spread of infection under certain conditions.

Prospective isolation is critical for understanding the cellular and molecular aspects of stem cell heterogeneity. Here, we identify the cell surface antigen CD9 as a positive marker that provides a simple alternative for hematopoietic stem cell isolation at high purity. Crucially, CD9 affords the capture of all hematopoietic stem cells in murine bone marrow in the absence of contaminating populations that lack authentic stem cell function. Using CD9 as a tool to subdivide hematopoietic stem-cell-containing populations, we provide evidence for heterogeneity at the cellular, functional, and molecular levels., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

This study was conducted to investigate the effect of vitrification of bovine metaphase-II (MII) oocytes on CD9 expression and fertilization capacity. Surviving vitrified/warmed oocytes were used to detect CD9 distribution (fluorescence microscopy), CD9 mRNA (qRT-PCR), and CD9 protein expression (Western blot), and to analyze in vitro fertilization rates (number of sperm bound to or that penetrated the oocytes) after removing the zona pellucida. Fresh oocytes acted as control. The experimental results showed that the vitrification/warming procedures significantly decreased CD9 expression at the mRNA and protein levels, and changed the CD9 distribution pattern in bovine oocytes. After fertilization in vitro, the average number of sperm binding and penetration of vitrified oocytes were significantly lower than those of the non-vitrified oocytes. In conclusion, vitrification of bovine oocytes caused a decrease in CD9 expression at the mRNA and protein levels, and an alteration of CD9 distribution pattern, which may have resulted in lowered fertilization capacity., (Copyright © 2013 Wiley Periodicals, Inc.)

Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells.

Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming.

Ovarian cancer is a gynecological cancer with a high death rate. We utilized global gene expression profiles of ovarian carcinomas obtained by complementary DNA (cDNA) microarray to identify ovarian cancer-specific proteins. CD9 was upregulated in ovarian carcinomas, and overexpression of the CD9 protein was detected in ovarian carcinomas by immunohistochemistry. CD9 was also overexpressed in several cancer cell lines, including ovarian cancer cells. In order to elucidate the biological significance of highly expressed CD9 in cancer cells, functional studies of CD9 were performed by ectopic expression, knockdown of CD9 using small interfering RNA (siRNA) and blockage of CD9 activity using the CD9-specific monoclonal antibody ALB6. Ectopic CD9 induced cell survival. In order to identify signaling pathways related to CD9, the gene expressions of CD9/SKOV3 cells were analyzed by cDNA microarray. Among the many upregulated genes, tumor necrosis factor (TNF)-α was induced in CD9/SKOV3 cells. The effect of overexpressed CD9 on the downstream signaling events of TNF-α was further investigated. In CD9/SKOV3 cells, the nuclear factor-kappaB (NF-κB)-signaling pathway was constitutively activated. Knockdown of CD9 by siRNA and blockage of CD9 activity by ALB6 in ovarian cancer cells demonstrated that constitutive activation of NF-κB is CD9 dependent and that CD9 is involved in anti-apoptosis. A CD9 functional study was performed in an ovarian cancer-xenograft mouse by injecting ALB6 into the peritoneum. ALB6 resulted in reduced tumor weight compared with that of control IgG(1). Collectively, these results demonstrate that CD9 functions as an oncogene and represents a target for the development of cancer-specific therapeutics.

It has been established that human dental pulp and periodontal ligament contain a population of mesenchymal stem cells (MSCs). However, the phenotypic analysis in terms of putative stem cell markers expressed by these stem cell populations is incomplete. It is relevant to understand whether stem cells derived from closely related tissues are programmed differently. The aim of the present study is to analyze whether these stem cells depict distinct characteristics by gaining insight into differences in their immunophenotype. Dental pulp and periodontal ligament tissue samples were obtained from extracted impacted wisdom teeth. Cell cultures were analyzed for surface and intracellular markers by indirect immunoflourescence. Detailed immunophenotype analysis was carried out by flow cytometry using relevant markers. The present study data shows dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) expressed embryonic stem (ES) cell markers Oct-4, Nanog and mesodermal marker Vimentin by indirect immunoflourescence. PDLSCs, however, had a weak expression of Nanog. Immunophenotyping revealed strong expression of MSC markers (CD73, CD90) in DPSCs and PDLSCs. Differences were observed in expression of stemness-related markers. DPSCs displayed increased percentages of SSEA4, CD13 and CD166 and decreased CD9 expression compared to PDLSCs. Both stem cells express common MSC markers, different levels of expression suggests there might be more than one stem cell population existing within these tissues which differ in their embryonic status, and DPSCs are a more primitive stem cell population in comparison to PDLSCs.