TSPAN6 reinforces the malignant progression of glioblastoma via interacting with CDK5RAP3 and regulating STAT3 signaling pathway.

Glioblastoma is the prevailing and highly malignant form of primary brain neoplasm with poor prognosis. Exosomes derived from glioblastoma cells act a vital role in malignant progression via regulating tumor microenvironment (TME), exosomal tetraspanin protein family members (TSPANs) are important actors of cell communication in TME. Among all the TSPANs, TSPAN6 exhibited predominantly higher expression levels in comparison to normal tissues. Meanwhile, glioblastoma patients with high level of TSPAN6 had shorter overall survival compared with low level of TSPAN6. Furthermore, TSPAN6 promoted the malignant progression of glioblastoma via promoting the proliferation and metastatic potential of glioblastoma cells. More interestingly, TSPAN6 overexpression in glioblastoma cells promoted the migration of vascular endothelial cell, and exosome secretion inhibitor reversed the migrative ability of vascular endothelial cells enhanced by TSPAN6 overexpressing glioblastoma cells, indicating that TSPAN6 might reinforce angiogenesis via exosomes in TME. Mechanistically, TSPAN6 enhanced the malignant progression of glioblastoma by interacting with CDK5RAP3 and regulating STAT3 signaling pathway. In addition, TSPAN6 overexpression in glioblastoma cells enhanced angiogenesis via regulating TME and STAT3 signaling pathway. Collectively, TSPAN6 has the potential to serve as both a therapeutic target and a prognostic biomarker for the treatment of glioblastoma.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells.

Bladder cancer aggressiveness is correlated with abnormal N-cadherin transmembrane glycoprotein expression. This protein is cleaved by the metalloprotease ADAM10 and the γ-secretase complex releasing a pro-angiogenic N-terminal fragment (NTF) and a proliferation-activating soluble C-terminal fragment (CTF2). Tetraspanin 15 (Tspan15) is identified as an ADAM10-interacting protein to induce selective N-cadherin cleavage. We first demonstrated, in invasive T24 bladder cancer cells, that N-cadherin was cleaved by ADAM10 generating NTF in the extracellular environment and leaving a membrane-anchored CTF1 fragment and that Tspan15 is required for ADAM10 to induce the selective N-cadherin cleavage. Targeting N-cadherin function in cancer is relevant to preventing tumor progression and metastases. For antitumor molecules to inhibit N-cadherin function, they should be complete and not cleaved. We first showed that the GW501516, an agonist of the nuclear receptor PPARß/δ, decreased Tspan15 and prevented N-cadherin cleavage thus decreasing NTF. Interestingly, the drug did not modify ADAM10 expression, which was important because it could limit side effects since ADAM10 cleaves numerous substrates. By targeting Tspan15 to block ADAM10 activity on N-cadherin, GW501516 could prevent NTF pro-tumoral effects and be a promising molecule to treat bladder cancer. More interestingly, it could optimize the effects of the N-cadherin antagonists those such as ADH-1 that target the N-cadherin ectodomain.

Molecular Determinants Involved in the Docking and Uptake of Tumor-Derived Extracellular Vesicles: Implications in Cancer.

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9.

CD81 and CD82 expressing tumor-infiltrating lymphocytes in the NSCLC tumor microenvironment play a crucial role in T-cell activation and cytokine production.

Introduction: To understand the immune system within the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC), it is crucial to elucidate the characteristics of molecules associated with T cell activation. Methods: We conducted an in-depth analysis using single-cell RNA sequencing data obtained from tissue samples of 19 NSCLC patients. T cells were classified based on the Tumor Proportion Score (TPS) within the tumor region, and molecular markers associated with activation and exhaustion were analyzed in T cells from high TPS areas. Results: Notably, tetraspanins CD81 and CD82, belonging to the tetraspanin protein family, were found to be expressed in activated T cells, particularly in cytotoxic T cells. These tetraspanins showed strong correlations with activation and exhaustion markers. In vitro experiments confirmed increased expression of CD81 and CD82 in IL-2-stimulated T cells. T cells were categorized into CD81highCD82high and CD81lowCD82low groups based on their expression levels, with CD81highCD82high T cells exhibiting elevated activation markers such as CD25 and CD69 compared to CD81lowCD82low T cells. This trend was consistent across CD3+, CD8+, and CD4+ T cell subsets. Moreover, CD81highCD82high T cells, when stimulated with anti-CD3, demonstrated enhanced secretion of cytokines such as IFN-γ, TNF-α, and IL-2, along with an increase in the proportion of memory T cells. Bulk RNA sequencing results after sorting CD81highCD82high and CD81lowCD82low T cells consistently supported the roles of CD81 and CD82. Experiments with overexpressed CD81 and CD82 showed increased cytotoxicity against target cells. Discussion: These findings highlight the multifaceted roles of CD81 and CD82 in T cell activation, cytokine production, memory subset accumulation, and target cell cytolysis. Therefore, these findings suggest the potential of CD81 and CD82 as promising candidates for co-stimulatory molecules in immune therapeutic strategies for cancer treatment within the intricate TME.

miR-378a-5p represses Barrett's esophagus cells proliferation, migration and invasion through targeting TSPAN8.

Barrett's esophagus (BE) belongs to a pathological phenomenon occurring in the esophagus, this paper intended to unveil the underlying function of miR-378a-5p and its target TSPAN8 in BE progression. GEO analysis was conducted to determine differentially expressed genes in BE samples. Non-dysplastic metaplasia BE samples, high-grade dysplastic BE samples and controls were collected from subjects. CP-A and CP-B cells were exposed to bile acids (BA) to mimic gastroesophageal reflux in BE cells. RT-qPCR as well as western blot were applied for verifying expressions of miR-378a-5p, TSPAN8, CDX2 and SOX9. CCK-8, wound scratch together with Transwell assays were exploited for ascertaining cell proliferation, migration as well as invasion. The targeted relationship of miR-378a-5p and TSPAN8 could be verified by correlation analysis, dual-luciferase reporter experiment, and rescue experiments. Through analyzing GSE26886 dataset, we screened the most abundantly expressed gene TSPAN8 in BE samples. miR-378a-5p was reduced whereas TSPAN8 was elevated in CP-A as well as CP-B cells after triggering with BA. Knocking down TSPAN8 could counteract BA-triggered enhancement in BE cell proliferation, migration along with invasion. miR-378a-5p could suppress BE cell proliferation, and migration along with invasion via targeting TSPAN8. In BE, miR-378a-5p targeted TSPAN8 to inhibit BE cell proliferation, and migration along invasion. miR-378a-5p deletion or elevation of TSPAN8 may be key point in regulating CDX2 and SOX9 levels, thereby promoting BE formation.

Tetraspanin CD82 Correlates with and May Regulate S100A7 Expression in Oral Cancer.

Many metastatic cancers with poor prognoses correlate to downregulated CD82, but exceptions exist. Understanding the context of this correlation is essential to CD82 as a prognostic biomarker and therapeutic target. Oral squamous cell carcinoma (OSCC) constitutes over 90% of oral cancer. We aimed to uncover the function and mechanism of CD82 in OSCC. We investigated CD82 in human OSCC cell lines, tissues, and healthy controls using the CRISPR-Cas9 gene knockout, transcriptomics, proteomics, etc. CD82 expression is elevated in CAL 27 cells. Knockout CD82 altered over 300 genes and proteins and inhibited cell migration. Furthermore, CD82 expression correlates with S100 proteins in CAL 27, CD82KO, SCC-25, and S-G cells and some OSCC tissues. The 37-50 kDa CD82 protein in CAL 27 cells is upregulated, glycosylated, and truncated. CD82 correlates with S100 proteins and may regulate their expression and cell migration. The truncated CD82 explains the invasive metastasis and poor outcome of the CAL 27 donor. OSCC with upregulated truncated CD82 and S100A7 may represent a distinct subtype with a poor prognosis. Differing alternatives from wild-type CD82 may elucidate the contradictory functions and pave the way for CD82 as a prognostic biomarker and therapeutic target.

Cutting Edge: The Tetraspanin CD53 Promotes CXCR4 Signaling and Bone Marrow Homing in B Cells.

B cell trafficking involves the coordinated activity of multiple adhesive and cytokine-receptor interactions, and the players in this process are not fully understood. In this study, we identified the tetraspanin CD53 as a critical regulator of both normal and malignant B cell trafficking. CXCL12 is a key chemokine in B cell homing to the bone marrow and secondary lymphoid organs, and both normal and malignant B cells from Cd53-/- mice have reduced migration toward CXCL12 in vitro, as well as impaired marrow homing in vivo. Using proximity ligation studies, we identified the CXCL12 receptor, CXCR4, as a novel, to our knowledge, CD53 binding partner. This interaction promotes receptor function, because Cd53-/- B cells display reduced signaling and internalization of CXCR4 in response to CXCL12. Together, our data suggest that CD53 interacts with CXCR4 on both normal and malignant B cells to promote CXCL12 signaling, receptor internalization, and marrow homing.

CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Molecular Regulation and Oncogenic Functions of TSPAN8.

Tetraspanins, a superfamily of small integral membrane proteins, are characterized by four transmembrane domains and conserved protein motifs that are configured into a unique molecular topology and structure in the plasma membrane. They act as key organizers of the plasma membrane, orchestrating the formation of specialized microdomains called "tetraspanin-enriched microdomains (TEMs)" or "tetraspanin nanodomains" that are essential for mediating diverse biological processes. TSPAN8 is one of the earliest identified tetraspanin members. It is known to interact with a wide range of molecular partners in different cellular contexts and regulate diverse molecular and cellular events at the plasma membrane, including cell adhesion, migration, invasion, signal transduction, and exosome biogenesis. The functions of cell-surface TSPAN8 are governed by ER targeting, modifications at the Golgi apparatus and dynamic trafficking. Intriguingly, limited evidence shows that TSPAN8 can translocate to the nucleus to act as a transcriptional regulator. The transcription of TSPAN8 is tightly regulated and restricted to defined cell lineages, where it can serve as a molecular marker of stem/progenitor cells in certain normal tissues as well as tumors. Importantly, the oncogenic roles of TSPAN8 in tumor development and cancer metastasis have gained prominence in recent decades. Here, we comprehensively review the current knowledge on the molecular characteristics and regulatory mechanisms defining TSPAN8 functions, and discuss the potential and significance of TSPAN8 as a biomarker and therapeutic target across various epithelial cancers.

Membrane organization by tetraspanins and galectins shapes lymphocyte function.

Immune receptors are not randomly distributed at the plasma membrane of lymphocytes but are segregated into specialized domains that function as platforms to initiate signalling, as exemplified by the B cell or T cell receptor complex and the immunological synapse. 'Membrane-organizing proteins' and, in particular, tetraspanins and galectins, are crucial for controlling the spatiotemporal organization of immune receptors and other signalling proteins. Deficiencies in specific tetraspanins and galectins result in impaired immune synapse formation, lymphocyte proliferation, antibody production and migration, which can lead to impaired immunity, tumour development and autoimmunity. In contrast to conventional ligand-receptor interactions, membrane organizers interact in cis (on the same cell) and modulate receptor clustering, receptor dynamics and intracellular signalling. New findings have uncovered their complex and dynamic nature, revealing shared binding partners and collaborative activity in determining the composition of membrane domains. Therefore, immune receptors should not be envisaged as independent entities and instead should be studied in the context of their spatial organization in the lymphocyte membrane. We advocate for a novel approach to study lymphocyte function by globally analysing the role of membrane organizers in the assembly of different membrane complexes and discuss opportunities to develop therapeutic approaches that act via the modulation of membrane organization.

TSPAN1 inhibits metastasis of nasopharyngeal carcinoma via suppressing NF-kB signaling.

Nasopharyngeal carcinoma (NPC) originates in the epithelial cells of the nasopharynx and is a common malignant tumor in southern China and Southeast Asia. Metastasis of NPC remains the main cause of death for NPC patients even though the tumor is sensitive to radiotherapy and chemotherapy. Here, we found that the transmembrane protein tetraspanin1 (TSPAN1) potently inhibited the in vitro migration and invasion, as well as, the in vivo metastasis of NPC cells via interacting with the IKBB protein. In addition, TSPAN1 was essential in preventing the overactivation of the NF-kB pathway in TSPAN1 overexpressing NPC cells. Furthermore, reduced TSPAN1 expression was associated with NPC metastasis and the poor prognosis of NPC patients. These results uncovered the suppressive role of TSPAN1 against NF-kB signaling in NPC cells for preventing NPC metastasis. Its therapeutic value warrants further investigation.

Tetraspanin 1 regulates papillary thyroid tumor growth and metastasis through c-Myc-mediated glycolysis.

Papillary thyroid cancer (PTC) is the most common form of thyroid cancer and is characterized by its tendency for lymphatic metastasis, leading to a poor prognosis. Tetraspanin 1 (TSPAN1) is a member of the tetra-transmembrane protein superfamily and has been implicated in tumorigenesis and cancer metastasis in various studies. However, the role of TSPAN1 in PTC tumor development remains unclear. In this study, we aimed to investigate the impact of TSPAN1 on PTC cell behavior. Our results demonstrate that knockdown of TSPAN1 inhibits PTC cell proliferation, migration, and invasion, while overexpression of TSPAN1 has the opposite effect. These findings suggest that TSPAN1 might play a role in the tumorigenesis and invasiveness of PTC. Mechanistically, we found that TSPAN1 activates the ERK pathway by increasing its phosphorylation, subsequently leading to upregulated expression of c-Myc. Additionally, we observed that TSPAN1-ERK-c-Myc axis activation promotes glycolytic activity in PTC cells, as evidenced by the upregulation of glycolytic genes such as LDHA. Taken together, our findings indicate that TSPAN1 acts as an oncogene in PTC by regulating glycolytic metabolism. This discovery highlights the potential of TSPAN1 as a promising therapeutic target for PTC treatment. Further research in this area could provide valuable insights into the development of targeted therapies for PTC patients.

TSPAN8 regulates EGFR/AKT pathway to enhance metastasis in gastric cancer.

BACKGROUND: Tetraspanin 8 (TSPAN8), a transmembrane glycoprotein, is implicated in various pathological conditions including human malignancies. However, the roles and underlying mechanisms of TSPAN8 in promoting gastric cancer(GC) progression are yet to be fully understood. METHODS AND RESULTS: Our study found that TSPAN8 expression was significantly elevated in GC tissues. We also observed a positive correlation between high TSPAN8 expression and various clinicopathological characteristics of GC, including tumor differentiation, invasion depth, lymph node metastasis, and clinical stage. Moreover, the elevated TSPAN8 expression was indicative of poor prognosis. Functionally, we observed that knockdown of TSPAN8 significantly attenuated while overexpression of TSPAN8 promoted GC cell migration and invasion. In vivo experiments, knockdown of TSPAN8 suppressed lung metastasis in nude mice. We further explored the underlying mechanisms of TSPAN8 and found that it regulated EGFR expression in GC cells by accelerating phosphorylation of EGFR and AKT. CONCLUSIONS: Our study reveals that TSPAN8 plays a significant role in promoting tumor metastasis by activating the EGFR/AKT pathway, indicating that it may serve as a promising therapeutic target of gastric cancer.

TET2-mediated mRNA demethylation regulates leukemia stem cell homing and self-renewal.

TET2 is recurrently mutated in acute myeloid leukemia (AML) and its deficiency promotes leukemogenesis (driven by aggressive oncogenic mutations) and enhances leukemia stem cell (LSC) self-renewal. However, the underlying cellular/molecular mechanisms have yet to be fully understood. Here, we show that Tet2 deficiency significantly facilitates leukemogenesis in various AML models (mediated by aggressive or less aggressive mutations) through promoting homing of LSCs into bone marrow (BM) niche to increase their self-renewal/proliferation. TET2 deficiency in AML blast cells increases expression of Tetraspanin 13 (TSPAN13) and thereby activates the CXCR4/CXCL12 signaling, leading to increased homing/migration of LSCs into BM niche. Mechanistically, TET2 deficiency results in the accumulation of methyl-5-cytosine (m5C) modification in TSPAN13 mRNA; YBX1 specifically recognizes the m5C modification and increases the stability and expression of TSPAN13 transcripts. Collectively, our studies reveal the functional importance of TET2 in leukemogenesis, leukemic blast cell migration/homing, and LSC self-renewal as an mRNA m5C demethylase.

TSPAN18 facilitates bone metastasis of prostate cancer by protecting STIM1 from TRIM32-mediated ubiquitination.

BACKGROUND: Bone metastasis is a principal cause of mortality in patients with prostate cancer (PCa). Increasing evidence indicates that high expression of stromal interaction molecule 1 (STIM1)-mediated store-operated calcium entry (SOCE) significantly activates the calcium (Ca2+) signaling pathway and is involved in multiple steps of bone metastasis in PCa. However, the regulatory mechanism and target therapy of STIM1 is poorly defined. METHODS: Liquid chromatography-mass spectrometry analysis was performed to identify tetraspanin 18 (TSPAN18) as a binding protein of STIM1. Co-IP assay was carried out to explore the mechanism by which TSPAN18 inhibits STIM1 degradation. The biological function of TSPAN18 in bone metastasis of PCa was further investigated in vitro and in vivo models. RESULT: We identified that STIM1 directly interacted with TSPAN18, and TSPAN18 competitively inhibited E3 ligase tripartite motif containing 32 (TRIM32)-mediated STIM1 ubiquitination and degradation, leading to increasing STIM1 protein stability. Furthermore, TSPAN18 significantly stimulated Ca2+ influx in an STIM1-dependent manner, and then markedly accelerated PCa cells migration and invasion in vitro and bone metastasis in vivo. Clinically, overexpression of TSPAN18 was positively associated with STIM1 protein expression, bone metastasis and poor prognosis in PCa. CONCLUSION: Taken together, this work discovers a novel STIM1 regulative mechanism that TSPAN18 protects STIM1 from TRIM32-mediated ubiquitination, and enhances bone metastasis of PCa by activating the STIM1-Ca2+ signaling axis, suggesting that TSPAN18 may be an attractive therapeutic target for blocking bone metastasis in PCa.

Comparative study of PRPH2 D2 loop mutants reveals divergent disease mechanism in rods and cones.

Mutations in the photoreceptor-specific tetraspanin gene peripherin-2 (PRPH2) lead to widely varying forms of retinal degeneration ranging from retinitis pigmentosa to macular dystrophy. Both inter- and intra-familial phenotypic heterogeneity has led to much interest in uncovering the complex pathogenic mechanisms of PRPH2-associated disease. Majority of disease-causing mutations in PRPH2 reside in the second intradiscal loop, wherein seven cysteines control protein folding and oligomerization. Here, we utilize knockin models to evaluate the role of three D2 loop cysteine mutants (Y141C, C213Y and C150S), alone or in combination. We elucidated how these mutations affect PRPH2 properties, including oligomerization and subcellular localization, and contribute to disease processes. Results from our structural, functional and molecular studies revealed that, in contrast to our understanding from prior investigations, rods are highly affected by PRPH2 mutations interfering with oligomerization and not merely by the haploinsufficiency associated with these mutations. On the other hand, cones are less affected by the toxicity of the mutant protein and significantly reduced protein levels, suggesting that knockdown therapeutic strategies may sustain cone functionality for a longer period. This observation provides useful data to guide and simplify the current development of effective therapeutic approaches for PRPH2-associated diseases that combine knockdown with high levels of gene supplementation needed to generate prolonged rod improvement.

The orf virus 129 protein can inhibit immune responses by interacting with host complement C1q binding protein in goat turbinate bone cells.

OBJECTIVE: Contagious ecthyma is a severe and highly contagious disease caused by an orf virus (ORFV). The virus is responsible for substantial economic losses in the goat industry and threatens humans. We previously determined the role of ORFV129 protein, one of the five ankyrin-repeat proteins coded by the orf genome, in suppressing the transcription of pro-inflammatory cytokines IL-6, IL-1ß and IFN-γ. In the present study, we identified 14 cellular proteins (complement C1q binding protein [C1QBP], MCM7, EIF5A, PKM, SLC6A, TSPAN6, ATP6AP2, GPS1, MMADHC, HSPB6, SLC35B1, MTF1, P3H4, and IL15RA) that interact with ORFV129 using a yeast two-hybrid system in goat turbinate bone cells (GFTCs). The interaction between ORFV129 and (C1QBP), an immune-related protein, was confirmed using immunofluorescence co-localization and co-immunoprecipitation assays. C1QBP overexpression inhibited ORFV replication, whereas the knockdown of C1QBP promoted ORFV replication in GFTCs. Furthermore, ORFV or ORFV129 increased C1QBP expression in GFTCs, indicated that ORFV129-C1QBP interaction might contribute to the ORFV-induced host immune process. In addition, our research showed that ORFV increased the expression of ORFV129, cytokine IL-6, IL-1ß and IFN-γ. C1QBP overexpression induced IFN-γ production and reduced IL-6 and IL-1ß production. Conversely, C1QBP knockdown induced IL-1ß production and reduced IFN-γ and IL-1ß production. Moreover, augmentation of ORFV129 expression enhanced the inhibition of the secretion of cytokines IL-6, IL-1ß, and IFN-γ induced by the altered expression of C1QBP. These findings suggest different downstream pathways might be involved in regulating different cytokines induced by ORFV129 expression in GFTCs.

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.

Surface protein profiling of milk and serum extracellular vesicles unveils body fluid-specific signatures.

Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease biomarkers. The promise of EV-based liquid biopsy resides in the identification of specific disease-associated EV signatures. Knowing the reference EV profile of a body fluid can facilitate the identification of such disease-associated EV-biomarkers. With this aim, we purified EVs from paired human milk and serum samples and used the MACSPlex bead-based flow-cytometry assay to capture EVs on bead-bound antibodies specific for a certain surface protein, followed by EV detection by the tetraspanins CD9, CD63, and CD81. Using this approach we identified body fluid-specific EV signatures, e.g. breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, as well as body fluid-specific markers associated to immune cells and stem cells. Interestingly, comparison of pan-tetraspanin detection (simultaneous CD9, CD63 and CD81 detection) and single tetraspanin detection (detection by CD9, CD63 or CD81) also unveiled body fluid-specific tetraspanin distributions on EVs. Moreover, certain EV surface proteins were associated with a specific tetraspanin distribution, which could be indicative of the biogenesis route of this EV subset. Altogether, the identified body fluid-specific EV profiles can contribute to study EV profile deviations in these fluids during disease processes.