RESUMO
A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.3) × 10(4) M(-1) s(-1) and 2.4 (±0.3) × 10(3) M(-1) s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities.
Assuntos
Proteínas ADAM/análise , Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Proteínas ADAM/química , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise , Análise de Regressão , SolubilidadeRESUMO
Natural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.
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Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Chaperonas Moleculares/farmacologia , Receptores de Células Matadoras Naturais/metabolismo , Evasão Tumoral/efeitos dos fármacos , Animais , Antígeno CD56/metabolismo , Antígeno CD56/fisiologia , Células Cultivadas , Exossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/fisiologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos SCID , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Receptores de IgG/metabolismo , Receptores de IgG/fisiologia , Receptores de Células Matadoras Naturais/agonistas , Receptores de Células Matadoras Naturais/antagonistas & inibidores , Solubilidade , Evasão Tumoral/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs.
Assuntos
Comunicação Celular , Extensões da Superfície Celular/metabolismo , Doença de Hodgkin/metabolismo , Antígeno Ki-1/metabolismo , Células de Reed-Sternberg/metabolismo , Vesículas Secretórias/metabolismo , Transdução de Sinais , Microambiente Tumoral , Biomarcadores Tumorais/metabolismo , Ligante CD30/metabolismo , Linhagem Celular Tumoral , Extensões da Superfície Celular/imunologia , Extensões da Superfície Celular/ultraestrutura , Microscopia Crioeletrônica , Eosinófilos/imunologia , Eosinófilos/metabolismo , Citometria de Fluxo , Granulócitos/imunologia , Granulócitos/metabolismo , Doença de Hodgkin/imunologia , Doença de Hodgkin/patologia , Humanos , Interleucina-8/metabolismo , Espectrometria de Massas , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Tamanho das Organelas , Células de Reed-Sternberg/imunologia , Células de Reed-Sternberg/ultraestrutura , Vesículas Secretórias/imunologia , Vesículas Secretórias/ultraestruturaRESUMO
NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody-derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re-directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell-mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti-tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD45+ immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti-tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.
Assuntos
Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/terapia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Células T Matadoras Naturais/imunologia , Transferência Adotiva , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígeno Carcinoembrionário/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteínas Ligadas por GPI/uso terapêutico , Células HEK293 , Humanos , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Lectinas Tipo C/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
Natural killer (NK) cells represent a key component of the innate immune system against cancer. Nevertheless, malignant diseases arise in immunocompetent individuals despite tumor immunosurveillance. Hodgkin lymphoma (HL) is characterized by CD30(+) tumor cells and a massive infiltration of immune effector cells in affected lymph nodes. The latter obviously fail to eliminate the malignant cell population. Here, we tested for functional NK cell defects in HL and suggest an improvement of NK function by therapeutic means. We demonstrate that peripheral NK cells (pNK) from patients with HL fail to eliminate HL cell lines in ex vivo killing assays. Impaired NK cell function correlated with elevated serum levels of soluble ligands for NK cell receptors NKp30 (BAG6/BAT3) and NKG2D (MICA), factors known to constrict NK cell function. In vitro, NK cell cytotoxicity could be restored by an NKG2D/NKp30-independent bispecific antibody construct (CD30xCD16A). It artificially links the tumor receptor CD30 with the cytotoxicity NK cell receptor CD16A. Moreover, we observed that NK cells from patients treated with this construct were generally activated and displayed a restored cytotoxicity against HL target cells. These data suggest that reversible suppression of NK cell activity contributes to immune evasion in HL and can be antagonized therapeutically.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Doença de Hodgkin/terapia , Células Matadoras Naturais/imunologia , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-HistoquímicaRESUMO
Direct interaction between T-cells exerts a major influence on tissue immunity and inflammation across multiple body sites including the human gut, which is highly enriched in 'unconventional' lymphocytes such as γδ T-cells. We previously reported that microbial activation of human Vγ9/Vδ2+ γδ T-cells in the presence of the mucosal damage-associated cytokine IL-15 confers the ability to promote epithelial barrier defence, specifically via induction of IL-22 expression in conventional CD4+ T-cells. In the current report, we assessed whether other cytokines enriched in the gut milieu also functionally influence microbe-responsive Vγ9/Vδ2 T-cells. When cultured in the presence of IL-21, Vγ9/Vδ2 T-cells acquired the ability to induce expression of the immunoregulatory cytokine IL-10 in both naïve and memory CD4+ T-cells, at levels surpassing those induced by monocytes or monocyte-derived DCs. These findings identify an unexpected influence of IL-21 on Vγ9/Vδ2 T-cell modulation of CD4+ T-cell responses. Further analyses suggested a possible role for CD30L and/or CD40L reverse signalling in mediating IL-10 induction by IL-21 conditioned Vγ9/Vδ2 T-cells. Our findings indicate that the local microenvironment exerts a profound influence on Vγ9/Vδ2 T-cell responses to microbial challenge, leading to induction of distinct functional profiles among CD4+ T-cells that may influence inflammatory events at mucosal surfaces. Targeting these novel pathways may offer therapeutic benefit in disorders such as inflammatory bowel disease.
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A disintegrin and metalloproteinase 17 (ADAM17) is significantly upregulated not only in malignant cells but also in the pro-inflammatory microenvironment of breast cancer. There, ADAM17 is critically involved in the processing of tumor-promoting proteins. Therefore, ADAM17 appears to be an attractive therapeutic target to address not only tumor cells but also the tumor-promoting environment. In a previous study, we generated a monoclonal anti-ADAM17 antibody (A300E). Although showing no complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity, the antibody was rapidly internalized by ADAM17-expressing cells and was able to transport a conjugated toxin into target cells. As a result, doxorubicin-coupled A300E or Pseudomonas exotoxin A-loaded A300E was able to kill ADAM17-expressing cells. This effect was strictly dependent on the presence of ADAM17 on the surface of target cells. As a proof of principle, both immunotoxins killed MDA-MB-231 breast cancer cells in an ADAM17-dependent manner. These data suggest that the use of anti-ADAM17 monoclonal antibodies as a carrier might be a promising new strategy for selective anti-cancer drug delivery.
Assuntos
Proteínas ADAM/imunologia , Antibióticos Antineoplásicos/administração & dosagem , Neoplasias da Mama/terapia , Doxorrubicina/administração & dosagem , Imunotoxinas/uso terapêutico , Proteínas ADAM/metabolismo , Proteína ADAM17 , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Neoplasias da Mama/metabolismo , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas do Sistema Complemento/imunologia , Feminino , Humanos , Terapia de Alvo MolecularRESUMO
Evasion of apoptosis is a hallmark of cancer cells. Inhibitor of apoptosis proteins (IAPs) act as endogenous inhibitors of programmed cell death and are overexpressed in several tumors including Hodgkin lymphoma (HL). Preclinical studies indicate antitumor activity of IAP antagonists and clinical studies in hematological malignancies are underway. Here, we investigate the impact of the small molecule IAP antagonist LCL161 on HL cell lines. Although the antagonist caused rapid degradation of cIAP1 leading to TNFα secretion, LCL161 did not promote apoptosis significantly. However, LCL161 induced expression of MICA and MICB, ligands for the activating immune receptor NKG2D, and enhanced the susceptibility of HL cells to NKG2D-dependent lysis by NK cells. MICA/B upregulation was dependent on activation of the DNA damage response upon LCL161 treatment. Taken together, we demonstrate a novel link between IAP inhibition, DNA damage and immune recognition.
Assuntos
Dano ao DNA/imunologia , Doença de Hodgkin/fisiopatologia , Imunidade Inata/imunologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/agonistas , Tiazóis/farmacologia , Regulação para Cima , Western Blotting , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/imunologia , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The interplay between dendritic cells (DCs) and natural killer (NK) cells directs adaptive immune responses. The molecular basis of the cross-talk is largely undefined. Here, we provide evidence for a contribution of CD30 (TNFRSF8) and its ligand CD30L (TNFSF8) expressed on NK cells and DCs, respectively. We demonstrate that CD30-mediated engagement of CD30L induced cytokine secretion from immature DCs via the mitogen-activated protein kinase pathway. Moreover, CD30L engagement promoted differentiation to mature DCs. On the contrary, the engagement of CD30 on NK cells resulted in an NF-κB-dependent release of TNF-α/IFN-γ. These data uncover a novel and unexpected role for CD30/CD30L that contributes to proinflammatory immune responses.
Assuntos
Ligante CD30/metabolismo , Células Dendríticas/metabolismo , Antígeno Ki-1/metabolismo , Células Matadoras Naturais/metabolismo , Transdução de Sinais , Ligante CD30/biossíntese , Células Dendríticas/citologia , Humanos , Antígeno Ki-1/biossíntese , Células Matadoras Naturais/citologiaAssuntos
Linfócitos B/metabolismo , Efeito Espectador/genética , Vesículas Extracelulares/química , Leucemia Linfocítica Crônica de Células B/genética , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Efeito Espectador/imunologia , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Neoplasias/imunologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/imunologia , Transporte de RNA , RNA Mensageiro/imunologia , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/imunologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Quinases da Família src/genética , Quinases da Família src/imunologiaRESUMO
Introduction: In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. Methods: We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Results: Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Conclusion: Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.
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BACKGROUND & AIMS: Liver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis. METHODS: Intracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor ß (TGFß) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model. RESULTS: In our study, we show that TGFß and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets' expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease. CONCLUSIONS: We provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.
Assuntos
Hepatite C Crônica , MicroRNAs/genética , Animais , Autofagia , Becaplermina/metabolismo , Células Estreladas do Fígado/patologia , Hepatite C Crônica/metabolismo , Hepatite C Crônica/patologia , Humanos , Cirrose Hepática/patologia , Camundongos , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
CD30, a member of the TNF receptor superfamily, is selectively expressed on a subset of activated lymphocytes and on malignant cells of certain lymphomas, such as classical Hodgkin Lymphoma (cHL), where it activates critical bystander cells in the tumor microenvironment. Therefore, it is not surprising that the CD30 antibody-drug conjugate Brentuximab Vedotin (BV) represents a powerful, FDA-approved treatment option for CD30+ hematological malignancies. However, BV also exerts a strong anti-cancer efficacy in many cases of diffuse large B cell lymphoma (DLBCL) with poor CD30 expression, even when lacking detectable CD30+ tumor cells. The mechanism remains enigmatic. Because CD30 is released on extracellular vesicles (EVs) from both, malignant and activated lymphocytes, we studied whether EV-associated CD30 might end up in CD30- tumor cells to provide binding sites for BV. Notably, CD30+ EVs bind to various DLBCL cell lines as well as to the FITC-labeled variant of the antibody-drug conjugate BV, thus potentially conferring the BV binding also to CD30- cells. Confocal microscopy and imaging cytometry studies revealed that BV binding and uptake depend on CD30+ EVs. Since BV is only toxic toward CD30- DLBCL cells when CD30+ EVs support its uptake, we conclude that EVs not only communicate within the tumor microenvironment but also influence cancer treatment. Ultimately, the CD30-based BV not only targets CD30+ tumor cell but also CD30- DLBCL cells in the presence of CD30+ EVs. Our study thus provides a feasible explanation for the clinical impact of BV in CD30- DLBCL and warrants confirming studies in animal models.
RESUMO
Cancer cells generally recruit and influence non-malignant immune cells to support the tumor growth. Classical Hodgkin lymphoma (cHL) is a good example because the affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (H-RS) cells, which are supported by a massive infiltrate of lymphocytes, fibroblasts, and innate immune cells. The transmembrane receptor CD30, which is selectively expressed on the H-RS cells, plays an important role, not only in cell stimulation and intercellular communication but also in tumor diagnosis and targeted tumor therapy. Different protein processing pathways influence its functionality. Depending on the conditions, the receptor is internalized or released. The release of CD30 occurs either as an intact molecule, embedded in the membrane of extracellular vesicles (EVs), or as a cleaved soluble ectodomain (sCD30). CD30 cleavage is predominantly catalyzed by ADAM10. The enzyme is catalytically active in cells as well as in EVs and gradually releases sCD30. Because the circulation contains no CD30+ donor cells, this mechanism explains that the cleaved ectodomain represents the predominant form of CD30 in the plasma of cHL patients. CD30 processing might influence the impact of CD30 antibody-drug conjugates, such as Brentuximab Vedotin (BV). Whereas, ADAM10-degraded CD30 impedes the BV efficacy, tumor-derived EVs load bystander cells with CD30 and generate new targets among supporter cells. This crossfire effect might contribute to the enormous clinical impact of BV, whereas the ADAM10-dependent cleavage to the mild systemic off-target effects of the treatment with BV.
Assuntos
Proteína ADAM10/imunologia , Comunicação Celular/imunologia , Doença de Hodgkin/imunologia , Antígeno Ki-1/imunologia , Vesículas Extracelulares/imunologia , HumanosRESUMO
The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.
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Proteína ADAM17 , Cisteína , Tiorredoxinas , Cisteína/metabolismo , Células HEK293 , Humanos , Conformação Molecular , Oxirredução , Compostos de Sulfidrila , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
CD30 is a transmembrane protein selectively overexpressed on many human lymphoma cells and therefore an interesting target for antibody-based immunotherapy. However, binding of therapeutic antibodies stimulates a juxtamembrane cleavage of CD30 leading to a loss of target antigen and an enhanced release of the soluble ectodomain of CD30 (sCD30). Here, we show that sCD30 binds to CD30 ligand (CD153)-expressing non-target cells. Because antibodies bind to sCD30, this results in unwanted antibody binding to these cells via sCD30 bridging. To overcome shedding-dependent damage of normal cells in CD30-specific immunotherapy, we analyzed the mechanism involved in the release. Shedding of CD30 can be enhanced by protein kinase C (PKC) activation, implicating the disintegrin metalloproteinase ADAM17 but not free cytoplasmic calcium. However, antibody-induced CD30 shedding is calcium dependent and PKC independent. This shedding involved the related metalloproteinase ADAM10 as shown by the use of the preferential ADAM10 inhibitor GI254023X and by an ADAM10-deficient cell line generated from embryonically lethal ADAM10(-/-) mouse. In coculture experiments, the antibody-induced transfer of sCD30 from the human Hodgkin's lymphoma cell line L540 to the CD30-negative but CD153-expressing human mast cell line HMC-1 was inhibited by GI254023X. These findings suggest that selective metalloproteinase inhibitors blocking antibody-induced shedding of target antigens could be of therapeutic value to increase the specificity and reduce side effects of immunotherapy with monoclonal antibodies.
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Proteínas ADAM/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Imunoterapia/métodos , Antígeno Ki-1/imunologia , Linfoma/imunologia , Linfoma/terapia , Proteínas de Membrana/antagonistas & inibidores , Proteínas ADAM/imunologia , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/imunologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Anticorpos/imunologia , Anticorpos/farmacologia , Ligante CD30/imunologia , Ligante CD30/metabolismo , Calcimicina/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Humanos , Antígeno Ki-1/metabolismo , Linfoma/enzimologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismoRESUMO
The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern's NanoSight NS300 or Particle Metrix' ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0-2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7-8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9-36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0-4.7) than measurements by NanoSight NS300 (% CV range: 5.4-10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5-134.3) compared to ZetaView (% DTEM range: 111.8-205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8-6.7; ZetaView: 1.4-7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Tumor-derived extracellular vesicles (EVs) induce pre-metastatic niche formation to promote metastasis. We isolated EVs from a highly-metastatic pancreatic cancer cell line and patient-derived primary cancer cells by ultracentrifugation. The protein content of EVs was analyzed by mass spectrometry. The effects of PDAC-derived EVs on natural kill (NK) cells were investigated by flow cytometry. The serum EVs' TGF-ß1 levels were quantified by ELISA. We found that integrins were enriched in PDAC-derived EVs. The expression of NKG2D, CD107a, TNF-α, and INF-γ in NK cells was significantly downregulated after co-culture with EVs. NK cells also exhibited decreased levels of CD71 and CD98, as well as impaired glucose uptake ability. In addition, NK cell cytotoxicity against pancreatic cancer stem cells was attenuated. Moreover, PDAC-derived EVs induced the phosphorylation of Smad2/3 in NK cells. Serum EVs' TGF-ß1 was significantly increased in PDAC patients. Our findings emphasize the immunosuppressive role of PDAC-derived EVs and provide new insights into our understanding of NK cell dysfunction regarding pre-metastatic niche formation in PDAC.
RESUMO
Extracellular vesicles released by tumor cells contribute to the reprogramming of the tumor microenvironment and interfere with hallmarks of cancer including metastasis. Notably, melanoma cell-derived EVs are able to establish a pre-metastatic niche in distant organs, or on the contrary, exert anti-tumor activity. However, molecular insights into how vesicles are selectively packaged with cargo defining their specific functions remain elusive. Methods: Here, we investigated the role of the chaperone Bcl2-associated anthogene 6 (BAG6, synonym Bat3) for the formation of pro- and anti-tumor EVs. EVs collected from wildtype cells and BAG6-deficient cells were characterized by mass spectrometry and RNAseq. Their tumorigenic potential was analyzed using the B-16V transplantation mouse melanoma model. Results: We demonstrate that EVs from B-16V cells inhibit lung metastasis associated with the mobilization of Ly6Clow patrolling monocytes. The formation of these anti-tumor-EVs was dependent on acetylation of p53 by the BAG6/CBP/p300-acetylase complex, followed by recruitment of components of the endosomal sorting complexes required for transport (ESCRT) via a P(S/T)AP double motif of BAG6. Genetic ablation of BAG6 and disruption of this pathway led to the release of a distinct EV subtype, which failed to suppress metastasis but recruited tumor-promoting neutrophils to the pre-metastatic niche. Conclusion: We conclude that the BAG6/CBP/p300-p53 axis is a key pathway directing EV cargo loading and thus a potential novel microenvironmental therapeutic target.
Assuntos
Exossomos/imunologia , Melanoma/imunologia , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Animais , Transformação Celular Neoplásica , Proteína p300 Associada a E1A/metabolismo , Vesículas Extracelulares/metabolismo , Células HEK293 , Humanos , Melanoma/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Microambiente TumoralRESUMO
Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed-Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.