Tetraspanin CD9 Research Articles

Predicting Cell Type-Specific Extracellular Vesicle Biology Using an Organism-Wide Single Cell Transcriptomic Atlas - Insights from the Tabula Muris

Background: Extracellular vesicles (EVs) like exosomes are functional nanoparticles traffcked between cells and found in every biofluid. An incomplete understanding of which cells, from which tissues, are traffcking EVs in vivo has limited our ability to use EVs as biomarkers and therapeutics. However, recent discoveries have linked EV secretion to expression of genes and proteins responsible for EV biogenesis and found as cargo, which suggests that emerging “cell atlas” datasets could be used to begin understanding EV biology at the level of the organism and possibly in rare cell populations. To explore this possibility, here we analyzed 67 genes that are directly implicated in EV biogenesis and secretion, or carried as cargo, in ~44,000 cells obtained from 117 cell populations of the Tabula Muris. Methods: We obtained normalized count (FPKM) data from 44,779 FACS-sorted cells described in the Tabula Muris. These data were freely obtained from the Tabula Muris web portal operated by the Chan-Zuckerberg Biohub. We selected 67 EV genes based on their direct role in the regulation of endosome biogenesis, protein sorting, secretion or cargo. These genes include the endosome sorting complexes required for transport (ESCRT), multiple Rab GTPases, tetraspanins and syndecans. To identify the most abundant EV genes, we plotted mean expression (FPKM) across all cells against the fraction of cells with gene expression > 50 FPKM. To measure gene expression variance, we calculated %CV for each gene within each cell population and across all cell populations. For gene co-expression analysis, we performed linear regressions comparing selected EV genes (Cd9, Cd63, Cd81, Sdc4 and Sdcbp) to all other genes, within each cell population. Results: We found that the most abundant EV cargo proteins (tetraspanins (Cd9, Cd63 and Cd81) and syndecans (Sdc4 and Sdcbp)) are also the most abundant EV genes expressed across all cell populations. However, the expression of these genes varied greatly among cell populations. For example, Cd81 is most abundant in brain glial populations and multiple endothelial cell populations. By contrast, Cd9 expression is highest in "barrier" cells that support innate immunity while Cd63, a classic marker of exosomes, is most abundantly expressed by stem cells. Expression variance analysis was used to compare the transcriptional behavior of these abundant EV genes (Cd9, Cd63, Cd81, Sdc4 and Sdcbp). We found that Cd63 is the most dynamic EV gene while others are more constitutively expressed. This finding may help explain why Cd63 is typically found either within the cytoplasm or outside of the cell on EVs, whereas Cd9 and Cd81 are abundant both on the cell surface and on EVs. Finally, we used EV gene co-expression analysis to define cell population-specific transcriptional networks. We discovered that EV gene co-expression networks are heavily dependent on the EV cargo gene examined with different cell populations driving clustering. Conclusions: Our analysis has provided at least four independent observations which advance our understanding of EV biology. These are that: 1) abundant EV cargo genes are differentially expressed among cell populations, 2) skeletal muscle stem cells display the highest expression of both Cd63 and Sdc4, 3) Cd81 may be a primary EV cargo traffcked in the brain whiand 4) that rare cell populations can secrete EVs and thereby influence tissue biology. This analysis is the first, to our knowledge, to predict tissue- and cell type-specific EV biology at the level of the organism. As such, we expect this resource to be the first of many valuable tools for predicting the endogenous impact of specific cell populations on EV function in health and disease. National Institutes on Aging (R01AG078859). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Screening of single nucleotide polymorphism in matrix metalloproteinase-2 (MMP2) and tetraspanin CD63 genes in Chlamydia trachomatis-infected tubal ectopic pregnancy patients.

Tubal ectopic pregnancy (EP) is a leading cause of maternal morbidity and mortality. Studies have suggested that infection-induced inflammatory responses are major risk factors for EP. The aim of the present study was to find an association between MMP2 and CD63 gene variants and risk of EP during Chlamydia trachomatis infection in an Indian population. Fallopian tube samples of 120 EP and 120 tubal ligation women were collected. C. trachomatis was detected by PCR. The genotyping of MMP2 (rs17859882 G/T, rs7201A/C) and CD63(rs2231464 C/T, rs376086542 A/G) gene variants was done by qualitative real-time PCR using allelic discrimination method (VIC- and FAM-labeled). The frequency of GG or GT genotype of MMP2 G/T polymorphism (rs17859882) was 66.6% in infected EP and 36.7% in uninfected EP and 22% in tubal ligation controls (P < 0.0001), while the frequency of AC or CC genotype of MMP2 A/C polymorphism (rs7201) was 66.6% in infected EP and 20.6% in uninfected EP and 13.5% in tubal ligation controls (P < 0.0001). The frequency of CT or TT genotype of CD63 C/T polymorphism (rs2231464) was 74% in infected EP and 21.8% in uninfected EP and 11.8% tubal ligation controls (P < 0.0001), while the frequency of AG or GG genotype of CD63 A/G polymorphism (rs376086542) was 48.1% in infected EP and 41.3% in uninfected EP and 18.6% tubal ligation controls (P < 0.0001). The present study revealed a strong association between the presence of gene variants MMP2 (rs17859882 G/T, rs7201A/C) and CD63 (rs2231464 C/T, rs376086542 A/G) and risk of tubal EP during C. trachomatis infection.

Synthetic Peptides with Genetic‐Codon‐Tailored Affinity for Assembling Tetraspanin CD81 at Cell Interfaces and Inhibiting Cancer Metastasis

AbstractProbing biomolecular interactions at cellular interfaces is crucial for understanding and interfering with life processes. Although affinity binders with site specificity for membrane proteins are unparalleled molecular tools, a high demand remains for novel multi‐functional ligands. In this study, a synthetic peptide (APQQ) with tight and specific binding to the untargeted extracellular loop of CD81 evolved from a genetically encoded peptide pool. With tailored affinity, APQQ flexibly accesses, site‐specifically binds, and forms a complex with CD81, enabling in‐situ tracking of the dynamics and activity of this protein in living cells, which has rarely been explored because of the lack of ligands. Furthermore, APQQ triggers the relocalization of CD81 from diffuse to densely clustered at cell junctions and modulates the interplay of membrane proteins at cellular interfaces. Motivated by these, efficient suppression of cancer cell migration, and inhibition of breast cancer metastasis were achieved in vivo.

Synthetic Peptides with Genetic-Codon-Tailored Affinity for Assembling Tetraspanin CD81 at Cell Interfaces and Inhibiting Cancer Metastasis.

Probing biomolecular interactions at cellular interfaces is crucial for understanding and interfering with life processes. Although affinity binders with site specificity for membrane proteins are unparalleled molecular tools, a high demand remains for novel multi-functional ligands. In this study, a synthetic peptide (APQQ) with tight and specific binding to the untargeted extracellular loop of CD81 evolved from a genetically encoded peptide pool. With tailored affinity, APQQ flexibly accesses, site-specifically binds, and forms a complex with CD81, enabling in-situ tracking of the dynamics and activity of this protein in living cells, which has rarely been explored because of the lack of ligands. Furthermore, APQQ triggers the relocalization of CD81 from diffuse to densely clustered at cell junctions and modulates the interplay of membrane proteins at cellular interfaces. Motivated by these, efficient suppression of cancer cell migration, and inhibition of breast cancer metastasis were achieved in vivo.

CD151 Maintains Endolysosomal Protein Quality to Inhibit Vascular Inflammation.

Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown. In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events. Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase. Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation.

Tetraspanin CD9 peptides for membrane disruptive on P. aeruginosa

The rapid development of multidrug-resistant strains and nosocomial P. aeruginosa infections pose a growing threat in the clinical setting. Tetraspanin CD9 peptides has demonstrated the antimicrobial activity against various gram-positive and -negative bacteria. Recently, CD9 peptides have shown anti-adhesion properties against P. aeruginosa isolates. In this study, we investigated the effect of CD9 peptides on the membrane of P. aeruginosa by transmission electron microscopy (TEM). The result shows the CD9 peptides cause disruption of the membrane of P. aeruginosa. In addition to the established antibacterial properties, this work reveals that tetraspanin CD9 peptides, a membrane-disrupting drug, could possibly be used as an additional treatment approach against P. aeruginosa. Future research should incorporate confocal microscopy to locate the bacterial matrix components and distinguish between living and dead P. aeruginosa upon the treatment with CD9 peptides.

Abstract 1537: CD63 acts as a key modulator of tumor progression and innate immune evasion in non-small cell lung cancer

Abstract Introduction: Lung cancer is a major reason for cancer-related mortality worldwide, causing more than two million cancer deaths per year. Over the last decade, new therapies have allowed long-term survival for patients even in advanced stages. However, only a minority of patients benefits from these therapies and patients often grow resistant over time. An important mechanism that contributes to cancer growth and the development of therapy resistances is the cross-talk between cancer cells and their tissue microenvironment, in particular fibroblasts and macrophages. In this context, we explored whether the tetraspanin CD63 - which is commonly found on intra- and extracellular membranes - stimulates cancer growth and mediates pro-tumorigenic functions of the tissue microenvironment in NSCLC. Methods: We analyzed patient tissue via immunohistochemistry and in-situ-hybridization. We deleted CD63 in cancer cell lines (A549, H810, LC19) and primary fibroblasts via CRISPR-Cas9. To assess phagocytosis, we used in vitro EdU assays and adaptive transfer models in vivo, and identified changes in cell signaling via mass spectrometry. We used an in vitro phagocytosis assay to determine the effect of CD63 on cancer cell phagocytosis through macrophages and analyzed the expression of M1 and M2-associated surface markers through flow cytometry upon co-culturing PBMC with cancer cells over one week. Results: In patient sections, cancer tissue did not only express more CD63 on the gene expression and protein level but a stronger CD63 expression also correlated with a denser macrophage infiltrate. Looking for pro-tumorigenic functions, deleting CD63 in three cancer cell lines reduced cancer cell growth both in vitro and in an adaptive transfer model in vivo. Additionally, deleting CD63 impaired the pro-tumorigenic focal adhesion signaling in primary fibroblasts. Concerning the interaction between cancer cells and macrophages, deleting CD63 reduced the macrophage-mediated phagocytosis of cancer cells and co-culturing primary monocytes with CD63-deficient lung cancer cells induced the expression of M1-associated surface markers on the PBMC. Conclusion: In conclusion, these results demonstrate that the tetraspanin CD63 drives NSCLC growth by stimulating cancer cell proliferation - in part by interfering with focal adhesion signaling - and facilitating immune evasion by preventing cancer cell phagocytosis through macrophages and inducing an immunosuppressive phenotype in macrophages. Citation Format: Tristan Lerbs, Lu Cui, Cristabelle De Souza, Reinhard Buettner, Gerlinde Wernig. CD63 acts as a key modulator of tumor progression and innate immune evasion in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1537.

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.

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. 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. 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. 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.

Weighted gene co-expression network analysis reveals immune evasion related genes in Echinococcus granulosus sensu stricto.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworm Echinococcus granulosus sensu lato (s.l). In the intermediate host, this disease is characterized by the growth of cysts in viscera such as liver and lungs, inside of which the parasite develops to the next infective stage known as protoscoleces. There are records that the infected viscera affect the development and morphology of E. granulosus s.l. protoscolex in hosts such as buffalo or humans. However, the molecular mechanisms that drive these differences remains unknown. Weighted gene co-expression network analysis (WGCNA) using a set of RNAseq data obtained from E. granulosus sensu stricto (s.s.) protoscoleces found in liver and lung cysts reveals 34 modules in protoscoleces of liver origin, of which 12 have differential co-expression from protoscoleces of lung origin. Three of these twelve modules contain hub genes related to immune evasion: tegument antigen, tegumental protein, ubiquitin hydrolase isozyme L3, COP9 signalosome complex subunit 3, tetraspanin CD9 antigen, and the methyl-CpG-binding protein Mbd2. Also, two of the twelve modules contain only hypothetical proteins with unknown orthology, which means that there are a group of unknown function proteins co-expressed inside the protoscolex of liver CE cyst origin. This is the first evidence of gene expression differences in protoscoleces from CE cysts found in different viscera, with co-expression networks that are exclusive to protoscoleces from liver CE cyst samples. This should be considered in the control strategies of CE, as intermediate hosts can harbor CE cysts in liver, lungs, or both organs simultaneously.

CD81 suppresses NF-κB signaling and is downregulated in hepatitis C virus expressing cells.

The tetraspanin CD81 is one of the main entry receptors for Hepatitis C virus, which is a major causative agent to develop liver cirrhosis and hepatocellular carcinoma (HCC). Here, we identify CD81 as one of few surface proteins that are downregulated in HCV expressing hepatoma cells, discovering a functional role of CD81 beyond mediating HCV entry. CD81 was downregulated at the mRNA level in hepatoma cells that replicate HCV. Kinetics of HCV expression were increased in CD81-knockout cells and accompanied by enhanced cellular growth. Furthermore, loss of CD81 compensated for inhibition of pro-survival TBK1-signaling in HCV expressing cells. Analysis of functional phenotypes that could be associated with pro-survival signaling revealed that CD81 is a negative regulator of NF-κB. Interaction of the NF-κB subunits p50 and p65 was increased in cells lacking CD81. Similarly, we witnessed an overall increase in the total levels of phosphorylated and cellular p65 upon CD81-knockout in hepatoma cells. Finally, translocation of p65 in CD81-negative hepatoma cells was markedly induced upon stimulation with TNFα or PMA. Altogether, CD81 emerges as a regulator of pro-survival NF-κB signaling. Considering the important and established role of NF-κB for HCV replication and tumorigenesis, the downregulation of CD81 by HCV and the associated increase in NF-κB signaling might be relevant for viral persistence and chronic infection.

Microscopic clusters feature the composition of biochemical tetraspanin-assemblies and constitute building-blocks of tetraspanin enriched domains

Biochemical approaches revealed that tetraspanins are multi-regulatory proteins forming a web, where they act in tetraspanin-enriched-microdomains (TEMs). A microscopic criterion differentiating between web and TEMs is lacking. Using super-resolution microcopy, we identify co-assemblies between the tetraspanins CD9 and CD81 and CD151 and CD81. CD9 assemblies contain as well the CD9/CD81-interaction partner EWI-2. Moreover, CD9 clusters are proximal to clusters of the CD81-interaction partner CD44 and CD81-/EWI-2-interacting ezrin–radixin–moesin proteins. Assemblies scatter unorganized across the cell membrane; yet, upon EWI-2 elevation, they agglomerate into densely packed arranged-crowds in a process independent from actin dynamics. In conclusion, microscopic clusters are equivalent to biochemical tetraspanin-assemblies, defining in their entirety the tetraspanin web. Cluster-agglomeration enriches tetraspanins, which makes agglomerations to a microscopic complement of TEMs. The microscopic classification of tetraspanin assemblies advances our understanding of this enigmatic protein family, whose members play roles in a plethora of cellular functions, diseases, and pathogen infections.

Integrin α2 is an early marker for osteoclast differentiation that contributes to key steps in osteoclastogenesis.

Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis. Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors. Our findings define integrin α2β1 as an early marker of OC differentiation.

Classifying tetraspanins: A universal system for numbering residues and a proposal for naming structural motifs and subfamilies

All tetraspanins have four transmembrane domains (TMs). The large extracellular loop (LEL) that connects the third and fourth TMs contains multiple secondary structures together with the family's signature Cys-Cys-Gly motif. These intriguing membrane proteins are involved in diverse and incompletely understood cellular processes including cell adhesion, tissue differentiation, immune cell maturation and host-parasite interactions. Here we present a classification system that accurately describes the position of each amino acid within its primary sequence based on both sequence and topological conservation of the TMs and LEL. This builds on the numbering systems that have been used in the G protein-coupled receptor (GPCR) field for nearly three decades and which have aided the understanding of GPCR structure/activity relationships and ligand interactions. The high-resolution structures of the tetraspanins CD81, CD9, CD53 and Tspan15 were used to validate the structural relevance of our new tetraspanin classification system. Modelling of all tetraspanin LELs highlighted flexibility in LEL disulfide bonding across the family and suggests that the structural arrangement of tetraspanin LELs is more complex than previously thought. We therefore propose a new subfamily naming system that addresses this added complexity and facilitates the systematic classification of human tetraspanins, shedding light on all structural motifs within the family. We anticipate that our universal tetraspanin classification system will enable progress in defining how sequence and structure inform function.

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

Single Cell Analysis of Genes Involved in Asymmetric Hematopoietic Stem Cell (HSC) Division in Chronic Myeloid Leukemia (CML) : Evidence of the Regulation of Tetraspanin CD63 during Leukemia Progression

Tyrosine kinase inhibitor (TKI) therapies have profoundly changed the natural history and survival of CML patients. However, the majority of the patients remain on long-lasting therapies and switch one TKI to another, either because of intolerance or resistance. All TKI cessation trials showed approximately 50% of leukemia recurrence rates even after several years of TKI therapies, suggesting the persistence of residual leukemic stem cells in patients in deep molecular response (DMR). The resistance and persistence of CML HSC are related to intrinsic (BCR-ABL independent survival) or extrinsic (leukemic niche ) mechanisms some of which have been extensively studied. However, there has been no data with regard to the role of asymmetric cell division pathways in the persistence and progression of CML stem cell clone. Indeed, During asymmetric division process of hematopoietic stem cells, some proteins are differently segregated between daughter HSCs such as endosome associated tetraspanin proteins (CD53, CD63) and mitochondria-associated fission regulator Dynamin related protein-1(DRP1). Similarly, it has been shown that asymmetric organelle inheritance can influence HSC decisions towards differentiation versus self-renewal. Interestingly, the tetraspanin CD63 is known to be involved in the quiescence of human and murine HSC via activation of TGF-beta pathway, CD63 hi cells exhibiting increased self-renewal potential after the asymmetric division with reduced differentiation potential. Materials and Methods We have analyzed the transcriptome performed in a cohort of CML patients in chronic phase (CP) and blast phase (BP) (GSE4170) as well as in a validation cohort of RNA-sequencing (GSE100026). Similarly, single cell transcriptome analysis of Lin- CD34+ CD38- CML progenitors (GSE76312) were included in the study evaluating the expression of genes involved in asymmetric cell division. We have then generated an asymmetric hematopoietic stem cell risk score using a panel of genes involved in the asymmetric division of HSCs and correlated these results with the transcriptome of the CML cohorts above. A pseudotime analysis was performed to evaluate single cell trajectories of the genes identified. Results To determine the potential role of asymmetric cell division markers in the progression of CML, we have analyzed the expression of several of these genes during CML progression in two independent transcriptome cohorts. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules which were found to be down-regulated during blast crisis as compared to chronic phase. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules down regulated during blast crisis as compared to chronic phase suggesting less of asymmetric properties during advanced phase of the disease. The down regulation in patients in blast crisis was confirmed at single cell level for the expression of SELL, CD63, NUMB, HK2 and LAMP2. CD63 expression has also been found to be of interest in CML chronic phase. Indeed, single cell neural network and deep learning in CP samples showed that the CD63 expression was the best asymmetric gene marker predicting a poor prognosis and this appeared to be induced by opposite regulation of HK2 and NUMB. In patients in CP-CML, the single cell trajectory reconstitution revealed the major regulatory role of CD63 highlighting a trajectory cluster implicating HSPB1, PIM2, ANXA5, LAMTOR1, CFL1, CD52, RAD52, MEIS1, PDIA3, all genes known to be involved also in hematopoietic malignancies such as myelodysplasia, AML and ALL. Conclusion We show here for the first time the identification of the expression of genes involved in the asymmetric division of CML HSC. Among these genes we identified the tetraspanin CD63 as being down-regulated during CML progression. The expression of the genes involved in the asymmetric HSC division should require further studies in CML, either to predict persistence of CML or progressive disease. Figure 1 : Single cell transcriptome of Lin- CD34+ CD38- CML progenitors in CP links CD63 expression to progressive disease. A/ Piechart of counts for chronic phase cells analyzed according to prognosis; B/ pseudotime transformation of single cell transcriptome according patient prognosis; C/ Pseudotime expression heatmap; D/ Pseudotime expression of CD63 in CP.

N-Glycosylation-dependent regulation of immune-specific tetraspanins CD37 and CD53

Tetraspanin proteins play an important role in many cellular processes as they are key organizers of different receptors on the plasma membrane. Most tetraspanins are highly glycosylated at their large extracellular loop; however, little is known about the function of tetraspanin glycosylation in immune cells. In this study we investigated the effects of glycosylation of CD37 and CD53, two tetraspanins important for cellular and humoral immunity. Broad and cell-specific repertoires of N-glycosylated CD37 and CD53 were observed in human B cells. We generated different glycosylation mutants of CD37 and CD53 and analyzed their localization, nanoscale plasma membrane organization, and partner protein interaction capacity. Abrogation of glycosylation in CD37 revealed the importance of this modification for CD37 surface expression, whereas surface expression of CD53 was unaffected by its glycosylation. Single-molecule dSTORM microscopy revealed that the nanoscale organization of CD53 was not dependent on glycosylation. CD37 interaction with its partner proteins CD53 and CD20 was affected by glycosylation in a localization-dependent way, whereas its interaction with IL-6Rα was independent of glycosylation. Surprisingly, glycosylation was found to inhibit the interaction between CD53 and its partner proteins CD45, CD20, and, to a lesser extent CD37. Together, our data show that glycosylation affects the interaction capacity of immune-specific tetraspanins CD37 and CD53, which adds another layer of regulation to immune membrane organization.

Dynamic Oscillation of Tetraspanin CD9 Orchestrates Engraftment and Mobilization of Human Hematopoietic Stem/Progenitor Cells

Background Trafficking of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche is pivotal for engraftment and mobilization during clinical transplantation, where both processes are controlled by highly orchestrated molecular programs. We previously identified the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis driving migration, adhesion and homing of human CD34+ HSPCs, but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remains unknown. Methods Umbilical cord blood (UCB) CD34+ cells were exposed to a CD9 neutralizing antibody ALB6 prior to infusion into sublethally irradiated NOD/SCID mice, with overall engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (B-lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitors) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was characterized by flow cytometry and compared to those before transplantation or after G-CSF mobilization. The oscillation pattern was then validated on HSPCs in pairwise BM, peripheral blood (PB) and G-CSF-mobilized peripheral blood (MPB) samples from healthy donors. Activity of integrin VLA-4 (an established binding partner of CD9) was evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay. Results A short exposure of UCB CD34+ cells to the CD9 neutralizing antibody markedly inhibited their BM engraftment by 64.2% ( P=0.003), reflected by the general decline in human CD45+ cell population. There was no bias in lymphoid/myeloid lineage outputs, suggesting CD9-mediated engraftment is not restricted to specific subsets of hematopoietic precursors. CD9 expression was drastically elevated on engrafted CD34+ cells in the BM from 9.6% to 91.4% ( P&amp;lt;0.001) compared to pre-transplantation. In contrast, CD9 expression was significantly downregulated on BM CD34+ cells from 73.4% to 33.5% ( P&amp;lt;0.001) upon G-CSF mobilization, with the decline observed in both primitive stem cells (CD34+CD38-) and committed progenitors (CD34+CD38+), indicating that the oscillation of CD9 is highly associated with HSPC trafficking. Concordantly, pairwise analysis of steady-state HSPCs in BM and PB from healthy donors revealed higher CD9 level in BM-residing CD34+ cells than the PB-circulating portion (46.1% vs. 26.5%, P=0.004; n=13). In addition, a significantly higher CD9 expression was observed on BM CD34+ cells (47.7%; n=16) than those in MPB (32.3%, n=25; P=0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure ( P=0.006), revealing a dynamic regulation. In normal BM, CD9+ HSPCs exhibited elevated VLA-4 activities with preferential binding to soluble VCAM-1 compared to the CD9- counterpart (51.4% vs. 25.9%, P&amp;lt;0.001; n=10). Superiority in VCAM-1 binding capacity was also witnessed in BM-residing CD34+ cells compared to MPB (37.7% vs. 4.1%, P=0.014), implicating that the influence of CD9 on HSPC trafficking may due to the alteration of integrin activities and downstream adhesive properties. Conclusion This study describes the dynamic regulation of CD9 during entry and exit of HSPCs to and from their BM niche via modulation of integrin activities. The findings extend our understandings on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.

Transcript CD81-215 may be a long noncoding RNA of stromal origin with tumor-promoting role in colon cancer.

The role of tetraspanin CD81 in malignant transformation is best studied in colorectal cancer, and it appears that other transcripts beside the fully coding mRNA may also be dysregulated in malignant cells. Recent data from a comprehensive pan-cancer transcriptome analysis demonstrated differential activity of two alternative CD81 gene promoters in malignant versus nonmalignant gut mucosa. The promoter active in gut mucosa gives rise to transcripts CD81-203 and CD81-213, while the promoter active in colon and rectal cancer gives rise to transcripts CD81-205 and CD81-215. Our study aimed to explore the biomarker potential of the transcripts from the alternative CD81 gene promoters in colon cancer, as well as to investigate their structure and potential function using in silico tools. The analysis of the transcripts'expression in several colon cell lines cultivated in 2D and 3D and a set of colon cancer and healthy gut mucosa samples by qPCR and RNA sequencing suggested their low expression and stromal origin. Expression patterns in tumor and nontumor tissue along with in silico data suppose that the transcript CD81-215 may be a noncoding RNA of stromal origin with possible involvement in signaling related to malignant transformation.

Tetraspanin CD81 Supports Cancer Stem Cell Function and Represents a Therapeutic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease associated with a poor outcome, necessitating aggressive therapies to achieve remission. A major contributing factor to treatment resistance and disease relapse is the presence of leukemic stem cells (LSC). Although challenging, targeting LSC constitutes an imperative approach to improve patient outcomes and limit side effects. In a previous study, we have uncovered a prognostic association between CD81 cell surface overexpression and worse outcome for AML patients. In the present work, we validated that CD81 overexpression of either protein or mRNA in AML blasts represents a specific prognostic marker for non-CBF AML with an extended cohort (n=150). We then confirmed our data using a publicly available dataset (BeatAML cohort). This association was also verified within NPM1 mutated patients, which was not associated to co- occurring FLT3-ITD mutations. When further investigating the underlying mechanisms related to CD81 overexpression and its impact on AML progression, we found out that CD81 expression was increased at relapse using paired diagnostic-relapsed samples (19% vs. 67%, P = 0.002). We demonstrated presence of CD81 within the LSC population, defined as CD34+/CD38-/CD90-/CD123+ blasts. Moreover, we showed that the CD81+ LSC fraction was higher at relapse compared to diagnosis (63% vs. 33%, P = 0.0001). In our cohort, non-CBF AML patients with higher level of CD81+LSC had worse outcome (RFS: P = 0.001, HR [95%CI] = 2.01 [1.16-3.48]; OS: P = 0.003; HR [95% CI] = 2.38 [1.32-4.29]. Next, we aimed to explore the potential impact of CD81 expression on LSC function, particularly focusing on its ability to serially engraft in immunocompromised mice (NSG). We observed a positive association between CD81 expression and an increased probability of positive engraftment, and a correlation with time-to-engraftment i.e., faster engraftment of xenografts from AML with high CD81 expression. Ex-vivo drug testing revealed primary AML cells as being more resistant to chemotherapy when engraftment was achieved. To investigate the mechanisms implied, we engineered either overexpression or knocked-down expression of CD81 in three different AML cell lines using cDNA transfection or shRNA, respectively. By means of these controlled CD81 expression models, we were able to demonstrate that CD81 overexpression increased AML cell adhesion, migration and invasion. Imaging by confocal microscopy exposed the development of membrane protrusions for CD81+ models. In vivo, higher CD81 expression increased blast homing, tumor burden and xenoengraftment efficiency. Moreover, we demonstrated that higher CD81 expression was linked to chemoresistance to anthracyclines and cytarabine, the two cornerstone drugs commonly used in AML. Finally, preclinical use of CD81 blocking antibody combined with chemotherapy in xenografts (AML cell line and patient-derived xenografts) resulted in longer relapse-free survival and reduced tumor aggressiveness. These results highlight the functional significance of CD81 overexpression in AML and its potential role as a biomarker for assessing LSC activity. Further investigations may improve this new avenue for targeted therapeutic intervention aimed at disrupting LSC function and improving treatment outcomes in AML patients.