RESUMO
Recent studies highlight the importance of baseline functional immunity for immune checkpoint blockade therapies. High-dimensional systemic immune profiling is performed in a cohort of non-small-cell lung cancer patients undergoing PD-L1/PD-1 blockade immunotherapy. Responders show high baseline myeloid phenotypic diversity in peripheral blood. To quantify it, we define a diversity index as a potential biomarker of response. This parameter correlates with elevated activated monocytic cells and decreased granulocytic phenotypes. High-throughput profiling of soluble factors in plasma identifies fractalkine (FKN), a chemokine involved in immune chemotaxis and adhesion, as a biomarker of response to immunotherapy that also correlates with myeloid cell diversity in human patients and murine models. Secreted FKN inhibits lung adenocarcinoma growth in vivo through a prominent contribution of systemic effector NK cells and increased tumor immune infiltration. FKN sensitizes murine lung cancer models refractory to anti-PD-1 treatment to immune checkpoint blockade immunotherapy. Importantly, recombinant FKN and tumor-expressed FKN are efficacious in delaying tumor growth in vivo locally and systemically, indicating a potential therapeutic use of FKN in combination with immunotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Humanos , Camundongos , Antígeno B7-H1/genética , Biomarcadores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/uso terapêutico , Neoplasias Pulmonares/genéticaRESUMO
BACKGROUND: Previous studies have shown that functional systemic immunity is required for the efficacy of PD-1/PD-L1 blockade immunotherapies in cancer. Hence, systemic reprogramming of immunosuppressive dysfunctional myeloid cells could overcome resistance to cancer immunotherapy. METHODS: Reprogramming of tumour-associated myeloid cells with oleuropein was studied by quantitative differential proteomics, phenotypic and functional assays in mice and lung cancer patients. Combinations of oleuropein and two different delivery methods of anti-PD-1 antibodies were tested in colorectal cancer tumour models and in immunotherapy-resistant lung cancer models. RESULTS: Oleuropein treatment reprogrammed monocytic and granulocytic myeloid-derived suppressor cells, and tumour-associated macrophages towards differentiation of immunostimulatory subsets. Oleuropein regulated major differentiation programmes associated to immune modulation in myeloid cells, which potentiated T cell responses and PD-1 blockade. PD-1 antibodies were delivered by two different strategies, either systemically or expressed within tumours using a self-amplifying RNA vector. Combination anti-PD-1 therapies with oleuropein increased tumour infiltration by immunostimulatory dendritic cells in draining lymph nodes, leading to systemic antitumour T cell responses. Potent therapeutic activities were achieved in colon cancer and lung cancer models resistant to immunotherapies, even leading to complete tumour regression. DISCUSSION: Oleuropein significantly improves the outcome of PD-1/PD-L1 blockade immunotherapy strategies by reprogramming myeloid cells.
Assuntos
Antígeno B7-H1 , Glucosídeos Iridoides , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Receptor de Morte Celular Programada 1 , Inibidores de Checkpoint Imunológico/farmacologia , Células Mieloides , Imunoterapia , Neoplasias Pulmonares/tratamento farmacológico , Microambiente TumoralRESUMO
CX3CL1 is one of the 50 up-to-date identified and characterized chemokines. While other chemokines are produced as small, secreted proteins, CX3CL1 (fractalkine) is synthetized as a transmembrane protein which also leads to a soluble form produced as a result of proteolytic cleavage. The membrane-bound protein and the soluble forms exhibit different biological functions. While the role of the fractalkine/CX3CR1 signaling axis was described in the nervous system and was also related to the migration of leukocytes to sites of inflammation, its actions are controversial in cancer progression and anti-tumor immunity. In the present review, we first describe the known biology of fractalkine concerning its action through its cognate receptor, but also its role in the activation of different integrins. The second part of this review is dedicated to its role in cancer where we discuss its role in anti-cancer or procarcinogenic activities.
Assuntos
Quimiocina CX3CL1 , Neoplasias , Humanos , Quimiocina CX3CL1/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Transdução de Sinais , Integrinas/metabolismo , Inflamação/metabolismoRESUMO
Since the introduction of the first anticancer treatments at the beginning of the 20th century, many different chemotherapeutics have been developed [...].
Assuntos
Antígeno B7-H1RESUMO
The application of BNT162b2 and mRNA-1273 vaccines against SARS-CoV-2 infection has constituted a determinant resource to control the COVID-19 pandemic. Since the beginning of 2021, millions of doses have been administered in several countries of North and South America and Europe. Many studies have confirmed the efficacy of these vaccines in a wide range of ages and in vulnerable groups of people against COVID-19. Nevertheless, the emergence and selection of new variants have led to a progressive decay in vaccine efficacy. Pfizer-BioNTech and Moderna developed updated bivalent vaccines-Comirnaty and Spikevax-to improve responses against the SARS-CoV-2 Omicron variants. Frequent booster doses with monovalent or bivalent mRNA vaccines, the emergence of some rare but serious adverse events and the activation of T-helper 17 responses suggest the need for improved mRNA vaccine formulations or the use of other types of vaccines. In this review, we discuss the advantages and limitations of mRNA vaccines targeting SARS-CoV-2 focusing on the most recent, related publications.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Vacina BNT162 , Pandemias , Vacinas de mRNA , Vacinas CombinadasRESUMO
Immunotherapies based on immune checkpoint blockade have shown remarkable clinical outcomes and durable responses in patients with many tumor types. Nevertheless, these therapies lack efficacy in most cancer patients, even causing severe adverse events in a small subset of patients, such as inflammatory disorders and hyper-progressive disease. To diminish the risk of developing serious toxicities, intratumor delivery of monoclonal antibodies could be a solution. Encouraging results have been shown in both preclinical and clinical studies. Thus, intratumor immunotherapy as a new strategy may retain efficacy while increasing safety. This approach is still an exploratory frontier in cancer research and opens up new possibilities for next-generation personalized medicine. Local intratumor delivery can be achieved through many means, but an attractive approach is the use of gene therapy vectors expressing mAbs inside the tumor mass. Here, we summarize basic, translational, and clinical results of intratumor mAb delivery, together with descriptions of non-viral and viral strategies for mAb delivery in preclinical and clinical development. Currently, this is an expanding research subject that will surely play a key role in the future of oncology.
Assuntos
Anticorpos Monoclonais , Neoplasias , Humanos , Anticorpos Monoclonais/uso terapêutico , Neoplasias/tratamento farmacológico , Imunoterapia/métodos , Medicina de PrecisãoRESUMO
Bispecific antibodies are a promising type of therapy for the treatment of cancer due to their ability to simultaneously inhibit different proteins playing a role in cancer progression. The development in lung cancer has been singularly intense because of the increasingly vast knowledge of the underlying molecular routes, in particular, in oncogene-driven tumors. In this review, we present the current landscape of bispecific antibodies for the treatment of lung cancer and discuss potential scenarios where the role of these therapeutics might expand in the near future.
Assuntos
Anticorpos Biespecíficos , Neoplasias Pulmonares , Humanos , Anticorpos Biespecíficos/uso terapêutico , Neoplasias Pulmonares/patologia , ImunoterapiaRESUMO
BACKGROUND: Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. METHODS: CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. RESULTS: CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. CONCLUSIONS: CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.
Assuntos
Antígenos CD/imunologia , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Antígenos CD/metabolismo , Antígeno B7-H1 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Receptor de Morte Celular Programada 1 , Linfócitos T , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
PD-L1/PD-1 blockade immunotherapy has changed the therapeutic approaches for the treatment of many cancers. Nevertheless, the mechanisms underlying its efficacy or treatment failure are still unclear. Proficient systemic immunity seems to be a prerequisite for efficacy, as recently shown in patients and in mouse models. It is widely accepted that expansion of anti-tumor CD8 T cell populations is principally responsible for anti-tumor responses. In contrast, the role of CD4 T cells has been less studied. Here we review and discuss the evidence supporting the contribution of CD4 T cells to anti-tumor immunity, especially recent advances linking CD4 T cell subsets to efficacious PD-L1/PD-1 blockade immunotherapy. We also discuss the role of CD4 T cell memory subsets present in peripheral blood before the start of immunotherapies, and their utility as predictors of response.
Assuntos
Antígeno B7-H1 , Imunoterapia , Neoplasias , Animais , Camundongos , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD4-Positivos , Fatores Imunológicos , Imunoterapia/métodos , Neoplasias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Subpopulações de Linfócitos TRESUMO
Vaccination, being able to prevent millions of cases of infectious diseases around the world every year, is the most effective medical intervention ever introduced. However, immunosenescence makes vaccines less effective in providing protection to older people. Although most studies explain that this is mainly due to the immunosenescence of T and B cells, the immunosenescence of innate immunity can also be a significant contributing factor. Alterations in function, number, subset, and distribution of blood neutrophils, monocytes, and natural killer and dendritic cells are detected in aging, thus potentially reducing the efficacy of vaccines in older individuals. In this paper, we focus on the immunosenescence of the innate blood immune cells. We discuss possible strategies to counteract the immunosenescence of innate immunity in order to improve the response to vaccination. In particular, we focus on advances in understanding the role and the development of new adjuvants, such as TLR agonists, considered a promising strategy to increase vaccination efficiency in older individuals.
Assuntos
Imunossenescência , Vacinas , Adjuvantes Imunológicos , Idoso , Envelhecimento , Humanos , Imunidade Inata , VacinaçãoRESUMO
The number of people that are 65 years old or older has been increasing due to the improvement in medicine and public health. However, this trend is not accompanied by an increase in quality of life, and this population is vulnerable to most illnesses, especially to infectious diseases. Vaccination is the best strategy to prevent this fact, but older people present a less efficient response, as their immune system is weaker due mainly to a phenomenon known as immunosenescence. The adaptive immune system is constituted by two types of lymphocytes, T and B cells, and the function and fitness of these cell populations are affected during ageing. Here, we review the impact of ageing on T and B cells and discuss the approaches that have been described or proposed to modulate and reverse the decline of the ageing adaptive immune system.
Assuntos
Imunossenescência , Imunidade Adaptativa , Idoso , Envelhecimento , Humanos , Qualidade de Vida , VacinaçãoRESUMO
BACKGROUND: The immunomodulation of the antitumor response driven by immunocheckpoint inhibitors (ICIs) such as PD-L1 (Programmed Death Ligand-1) monoclonal antibody (α-PD-L1) have shown relevant clinical outcomes in a subset of patients. This fact has led to the search for rational combinations with other therapeutic agents such as Doxorubicin (Dox), which cytotoxicity involves an immune activation that may enhance ICI response. Therefore, this study aims to evaluate the combination of chemotherapy and ICI by developing Dox Immunoliposomes functionalized with monovalent-variable fragments (Fab') of α-PD-L1. RESULTS: Immunoliposomes were assayed in vitro and in vivo in a B16 OVA melanoma murine cell line over-expressing PD-L1. Here, immune system activation in tumor, spleen and lymph nodes, together with the antitumor efficacy were evaluated. Results showed that immunoliposomes bound specifically to PD-L1+ cells, yielding higher cell interaction and Dox internalization, and decreasing up to 30-fold the IC50, compared to conventional liposomes. This mechanism supported a higher in vivo response. Indeed, immunoliposomes promoted full tumor regression in 20% of mice and increased in 1 month the survival rate. This formulation was the only treatment able to induce significant (p < 0.01) increase of activated tumor specific cytotoxic T lymphocytes at the tumor site. CONCLUSION: PD-L1 targeted liposomes encapsulating Dox have proved to be a rational combination able to enhance the modulation of the immune system by blocking PD-L1 and selectively internalizing Dox, thus successfully providing a dual activity offered by both, chemo and immune therapeutic strategies.
Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Doxorrubicina/farmacologia , Imunidade/efeitos dos fármacos , Lipossomos/imunologia , Melanoma/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Liberação Controlada de Fármacos , Tratamento Farmacológico , Feminino , Imunoterapia/métodos , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Lymphocyte activation gene 3 (LAG-3) is a cell surface inhibitory receptor with multiple biological activities over T cell activation and effector functions. LAG-3 plays a regulatory role in immunity and emerged some time ago as an inhibitory immune checkpoint molecule comparable to PD-1 and CTLA-4 and a potential target for enhancing anti-cancer immune responses. LAG-3 is the third inhibitory receptor to be exploited in human anti-cancer immunotherapies, and it is considered a potential next-generation cancer immunotherapy target in human therapy, right next to PD-1 and CTLA-4. Unlike PD-1 and CTLA-4, the exact mechanisms of action of LAG-3 and its relationship with other immune checkpoint molecules remain poorly understood. This is partly caused by the presence of non-conventional signaling motifs in its intracellular domain that are different from other conventional immunoregulatory signaling motifs but with similar inhibitory activities. Here we summarize the current understanding of LAG-3 signaling and its role in LAG-3 functions, from its mechanisms of action to clinical applications.
Assuntos
Antígenos CD/metabolismo , Antígenos CD/fisiologia , Transdução de Sinais/fisiologia , Humanos , Imunoterapia , Ativação Linfocitária , Neoplasias/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/imunologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Along with the positioning of immunotherapy as a preferential treatment for a wide variety of neoplasms, a new pattern of response consisting in a sudden acceleration of tumor growth has been described. This phenomenon has received the name of "hyperprogressive disease", and several definitions have been proposed for its identification, most of them relying on radiological criteria. However, due to the fact that the cellular and molecular mechanisms have not been elucidated yet, there is still some debate regarding whether this fast progression is induced by immunotherapy or only reflects the natural course of some highly aggressive neoplasms. Moreover, contradictory results of trials including patients with different cancer types suggest that both the incidence, the associated factors and the implications regarding prognosis might differ depending on tumor histology. This article intends to review the main publications regarding this matter and critically approach the most controversial aspects.
Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia , Neoplasias , Humanos , Neoplasias/diagnóstico , Neoplasias/imunologia , Neoplasias/terapia , PrognósticoRESUMO
Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Expressão Gênica , Vetores Genéticos/genética , Neoplasias/genética , Neoplasias/imunologia , Vírus de RNA/genética , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Injeções Intralesionais , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/genética , Vírus da Floresta de Semliki/genética , Taxa de Sobrevida , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Carga TumoralRESUMO
The development of cancer immunotherapy in the last decade has followed a vertiginous rhythm. Nowadays, immune checkpoint inhibitors (ICI) which include anti-CTLA4, anti-PD-1 and anti-PD-L1 antibodies are in clinical use for the treatment of numerous cancers. However, approximately only a third of the patients benefit from ICI therapies. Many efforts have been made for the identification of biomarkers allowing patient stratification into potential responders and progressors before the start of ICI therapies or for monitoring responses during treatment. While much attention is centered on biomarkers from the tumor microenvironment, in many cases biopsies are not available. The identification of systemic immune cell subsets that correlate with responses could provide promising biomarkers. Some of them have been reported to influence the response to ICI therapies, such as proliferation and activation status of CD8 and CD4 T cells, the expression of immune checkpoints in peripheral blood cells and the relative numbers of immunosuppressive cells such as regulatory T cells and myeloid-derived suppressor cells. In addition, the profile of soluble factors in plasma samples could be associated to response or tumor progression. Here we will review the cellular subsets associated to response or progression in different studies and discuss their accuracy in diagnosis.
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Biomarcadores , Células Sanguíneas , Inibidores de Checkpoint Imunológico/uso terapêutico , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Antígeno CTLA-4 , Humanos , Imunoterapia , Células Matadoras Naturais , Macrófagos , Monócitos , Células Supressoras Mieloides/metabolismo , Neoplasias/tratamento farmacológico , Microambiente Tumoral/imunologiaRESUMO
The use of monoclonal antibodies targeting PD-1/PD-L1 axis completely changed anticancer treatment strategies. However, despite the significant improvement in overall survival and progression-free survival of patients undergoing these immunotherapy treatments, the only clinically accepted biomarker with some prediction capabilities for the outcome of the treatment is PD-L1 expression in tumor biopsies. Nevertheless, even when having PD-L1-positive tumors, numerous patients do not respond to these treatments. Considering the high cost of these therapies and the risk of immune-related adverse events during therapy, it is necessary to identify additional biomarkers that would facilitate stratifying patients in potential responders and non-responders before the start of immunotherapies. Here, we review the utility of PD-L1 expression not only in tumor cells but in immune system cells and their influence on the antitumor activity of immune cell subsets.
Assuntos
Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Neoplasias/terapia , Animais , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Imunoterapia/efeitos adversos , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismoRESUMO
PD-L1 tumor expression is a widely used biomarker for patient stratification in PD-L1/PD-1 blockade anticancer therapies, particularly for lung cancer. However, the reliability of this marker is still under debate. Moreover, PD-L1 is widely expressed by many immune cell types, and little is known on the relevance of systemic PD-L1⺠cells for responses to immune checkpoint blockade. We present two clinical cases of patients with non-small cell lung cancer (NSCLC) and PD-L1-negative tumors treated with atezolizumab that showed either objective responses or progression. These patients showed major differences in the distribution of PD-L1 expression within systemic immune cells. Based on these results, an exploratory study was carried out with 32 cases of NSCLC patients undergoing PD-L1/PD-1 blockade therapies, to compare PD-L1 expression profiles and their relationships with clinical outcomes. Significant differences in the percentage of PD-L1⺠CD11b⺠myeloid cell populations were found between objective responders and non-responders. Patients with percentages of PD-L1⺠CD11b⺠cells above 30% before the start of immunotherapy showed response rates of 50%, and 70% when combined with memory CD4 T cell profiling. These findings indicate that quantification of systemic PD-L1⺠myeloid cell subsets could provide a simple biomarker for patient stratification, even if biopsies are scored as PD-L1 null.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Imunoterapia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismoRESUMO
AIM: To evaluate the radiopotentiation of enzalutamide in human prostate cancer cells. BACKGROUND: While radiotherapy is the first line of treatment for prostate cancer, androgen blockade therapies are demonstrating significant survival benefit as monotherapies. As androgen blockade can cause cell death by apoptosis, it is likely that androgen blockade will potentiate the cytotoxic activities of radiotherapy. MATERIALS AND METHODS: Here, we tested the potential synergistic effects of these two treatments over two human metastatic prostate cancer cells by real-time cell analysis (RTCA), androgen-sensitive LNCaP cells (Lymph Node Carcinoma of the Prostate) and androgen-independent PC-3. Both cell lines were highly resistant to high doses of radiotherapy. RESULTS: A pre-treatment of LNCaP cells with IC50 concentrations of enzalutamide significantly sensitized them to radiotherapy through enhanced apoptosis. In contrast, enzalutamide resistant PC-3 cells were not sensitized to radiotherapy by androgen blockade. CONCLUSIONS: These results provide evidence that the enzalutamide/radiotherapy combination could maximize therapeutic responses in patients with enzalutamide-sensitive prostate cancer.
RESUMO
Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.