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MOTIVATION: Cytometry comprises powerful techniques for analyzing the cell heterogeneity of a biological sample by examining the expression of protein markers. These technologies impact especially the field of oncoimmunology, where cell identification is essential to analyze the tumor microenvironment. Several classification tools have been developed for the annotation of cytometry datasets, which include supervised tools that require a training set as a reference (i.e. reference-based) and semisupervised tools based on the manual definition of a marker table. The latter is closer to the traditional annotation of cytometry data based on manual gating. However, they require the manual definition of a marker table that cannot be extracted automatically in a reference-based fashion. Therefore, we are lacking methods that allow both classification approaches while maintaining the high biological interpretability given by the marker table. RESULTS: We present a new tool called GateMeClass (Gate Mining and Classification) which overcomes the limitation of the current methods of classification of cytometry data allowing both semisupervised and supervised annotation based on a marker table that can be defined manually or extracted from an external annotated dataset. We measured the accuracy of GateMeClass for annotating three well-established benchmark mass cytometry datasets and one flow cytometry dataset. The performance of GateMeClass is comparable to reference-based methods and marker table-based techniques, offering greater flexibility and rapid execution times. AVAILABILITY AND IMPLEMENTATION: GateMeClass is implemented in R language and is publicly available at https://github.com/simo1c/GateMeClass.
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Minería de Datos , Citometría de Flujo , Citometría de Flujo/métodos , Minería de Datos/métodos , Humanos , Programas Informáticos , Algoritmos , Microambiente TumoralRESUMEN
OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients' tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Fenotipo , Semaforina-3A , Animales , Humanos , Ratones , Orientación del Axón/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Semaforina-3A/metabolismo , Semaforina-3A/genética , Transducción de SeñalRESUMEN
Graft-vs-host-disease (GvHD) is currently the main complication of allogeneic hematopoietic stem cell transplantation. Mortality and morbidity rates are particularly high, especially in steroid-refractory acute GvHD (aGvHD). Immune regulatory human bone marrow mesenchymal stromal cells (hMB-MSCs) represent a therapeutic approach to address this issue. Unfortunately, their effect is hardly predictable in vivo due to several variables, that is, MSC tissue origin, concentration, dose number, administration route and timing, and inflammatory status of the recipient. Interestingly, human bone marrow MSC-derived extracellular vesicles (hBM-MSC-EVs) display many of the hBM-MSC immunoregulatory properties due to their content in paracrine factors that greatly varies according to the collection method. In this study, we focused on the immunological characterization of hBM-MSC-EVs on their capability of inducing regulatory T-cells (T-regs) both in vitro and in a xenograft mouse model of aGvHD. We correlated these data with the aGvHD incidence and degree following hBM-MSC-EV intravenous administration. Thus, we first quantified the EV immunomodulation in vitro in terms of EV immunomodulatory functional unit (EV-IFU), that is, the lowest concentration of EVs leading in vitro to at least threefold increase of the T-regs compared with controls. Second, we established the EV therapeutic dose in vivo (EV-TD) corresponding to 10-fold the in vitro EV-IFU. According to this approach, we observed a significant improvement of both mouse survival and control of aGvHD onset and progression. This study confirms that EVs may represent an alternative to whole MSCs for aGvHD prevention, once the effective dose is reproducibly identified according to EV-IFU and EV-TD definition.
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Vesículas Extracelulares/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Células Madre Mesenquimatosas/metabolismo , Animales , Femenino , Humanos , RatonesRESUMEN
Heterozygous mutations in the PHOX2B gene are causative of congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by defective autonomic control of breathing due to the impaired differentiation of neural crest cells. Among PHOX2B mutations, polyalanine (polyAla) expansions are almost exclusively associated with isolated CCHS, whereas frameshift variants, although less frequent, are often more severe than polyAla expansions and identified in syndromic CCHS. This article provides a complete review of all the frameshift mutations identified in cases of isolated and syndromic CCHS reported in the literature as well as those identified by us and not yet published. These were considered in terms of both their structure, whether the underlying indels induced frameshifts of either 1 or 2 steps ("frame 2" and "frame 3" mutations respectively), and clinical associations. Furthermore, we evaluated the structural and functional effects of one "frame 3" mutation identified in a patient with isolated CCHS, and one "frame 2" mutation identified in a patient with syndromic CCHS, also affected with Hirschsprung's disease and neuroblastoma. The data thus obtained confirm that the type of translational frame affects the severity of the transcriptional dysfunction and the predisposition to isolated or syndromic CCHS.
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Mutación del Sistema de Lectura/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Hipoventilación/congénito , Apnea Central del Sueño/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Hipoventilación/genética , Hipoventilación/metabolismo , Microscopía Fluorescente , Mutación , Apnea Central del Sueño/metabolismoRESUMEN
HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.
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Secuencia de Bases , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Enfermedad de Hirschsprung/metabolismo , Proteínas de Homeodominio/metabolismo , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-ret/biosíntesis , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Transcripción Genética , Línea Celular Tumoral , Femenino , Enfermedad de Hirschsprung/genética , Enfermedad de Hirschsprung/patología , Proteínas de Homeodominio/genética , Humanos , Masculino , Péptidos/genética , Proteínas Proto-Oncogénicas c-ret/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: High interleukin-8 (IL-8) levels have been linked to poor prognosis in lung cancer, but conclusive data are lacking. MATERIALS AND METHODS: A comprehensive search was conducted on April 1st, 2023, from electronic databases, focusing on studies with IL-8 expression evaluations and the availability of hazard ratio (HR) and 95% confidence intervals (CI) for overall survival (OS), progression-free survival (PFS) and disease-free survival (DFS) or adequate data for their estimation. Then, we examined IL-8 and CXCR1 RNA-seq data from The Cancer Genome Atlas (TCGA) dataset, and we correlated these data with OS. RESULTS: Among 2655 produced records, 10 manuscripts involving both non-small cell lung cancer and small cell lung cancer, were included in the analysis. Two manuscripts and one study included two and three different cohorts, respectively, for a total of 14 cohorts of patients. Overall, 4 cohorts evaluated IL-8 levels in patients treated with chemotherapy, 3 cohorts immunotherapy, 2 cohorts surgical patients and 4 cohorts other treatments; 1 cohort was removed, as the type of treatments was lacking. The 12 cohorts included in the OS analysis revealed that patients with high IL-8 levels have a lower OS probability, as compared to patients with low IL-8 levels (HR=1.75, 95 % CI 1.36-2.26). No significant difference between patients with high and low IL-8 levels was observed in the 8 cohorts available for PFS analysis. Sensitivity analysis according to treatment revealed significant PFS and OS differences for patients treated with chemotherapy or immunotherapy. Analysis of RNA-seq data from TCGA, confirmed the correlation between high IL-8 and CXCR1 expression and worse OS in patients with resected lung cancer. CONCLUSION: To the best of our knowledge, this study represents the first meta-analysis demonstrating a negative prognostic impact of high IL-8 level in lung cancer, particularly in patients treated with chemotherapy and/or immunotherapy.
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Biología Computacional , Interleucina-8 , Neoplasias Pulmonares , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Pronóstico , Biología Computacional/métodos , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patologíaRESUMEN
Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.
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Linfoma de Células del Manto , Humanos , Adulto , Linfoma de Células del Manto/patología , Factor de Transcripción STAT5/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
Tumour microenvironment is a complex ecosystem in which myeloid cells are the most abundant immune elements. This cell compartment is composed by different cell types, including neutrophils, macrophages, dendritic cells, and monocytes but also unexpected cell populations with immunosuppressive and pro-tumour roles. Indeed, the release of tumour-derived factors influences physiological haematopoiesis producing unconventional cells with immunosuppressive and tolerogenic functions such as myeloid-derived suppressor cells. These pro-tumour myeloid cell populations not only support immune escape directly but also assist tumour invasion trough non-immunological activities. It is therefore not surprising that these cell subsets considerably impact in tumour progression and cancer therapy resistance, including immunotherapy, and are being investigated as potential targets for developing a new era of cancer therapy. In this review, we discuss emerging strategies able to modulate the functional activity of these tumour-supporting myeloid cells subverting their accumulation, recruitment, survival, and functions. These innovative approaches will help develop innovative, or improve existing, cancer treatments.
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Células Supresoras de Origen Mieloide , Neoplasias , Humanos , Ecosistema , Células Mieloides , Inmunoterapia , Macrófagos , Microambiente TumoralRESUMEN
Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8+ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8+ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration.
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Trampas Extracelulares , Neoplasias Pancreáticas , Humanos , Animales , Ratones , Linfocitos T CD8-positivos , Trampas Extracelulares/metabolismo , Arginasa/metabolismo , Inmunoterapia , Neoplasias Pancreáticas/terapia , Anticuerpos Monoclonales/farmacología , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
The advent of immune checkpoint inhibitors (ICIs), for instance, programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockers, has greatly improved the outcome of patients affected by non-small cell lung cancer (NSCLC). However, most NSCLC patients either do not respond to ICI monotherapy or develop resistance to it after an initial response. Therefore, the identification of biomarkers for predicting the response of patients to ICI monotherapy represents an urgent issue. Great efforts are currently dedicated toward identifying blood-based biomarkers to predict responses to ICI monotherapy. In this study, more commonly utilized blood-based biomarkers, such as the neutrophil-to-lymphocyte ratio (NLR) and the lung immune prognostic index (LIPI) score, as well as the frequency/number and activation status of various types of circulating innate immune cell populations, were evaluated in NSCLC patients at baseline before therapy initiation. The data indicated that, among all the parameters tested, low plasmacytoid dendritic cell (pDC), slan+-monocyte and natural killer cell counts, as well as a high LIPI score and elevated PD-L1 expression levels on type 1 conventional DCs (cDC1s), were independently correlated with a negative response to ICI therapy in NSCLC patients. The results from this study suggest that the evaluation of innate immune cell numbers and phenotypes may provide novel and promising predictive biomarkers for ICI monotherapy in NSCLC patients.
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Cancer cells favor the generation of myeloid cells with immunosuppressive and inflammatory features, including myeloid-derived suppressor cells (MDSCs), which support tumor progression. The anti-apoptotic molecule, cellular FLICE (FADD-like interleukin-1ß-converting enzyme)-inhibitory protein (c-FLIP), which acts as an important modulator of caspase-8, is required for the development and function of monocytic (M)-MDSCs. Here, we assessed the effect of immune checkpoint inhibitor (ICI) therapy on systemic immunological landscape, including FLIP-expressing MDSCs, in non-small cell lung cancer (NSCLC) patients. Longitudinal changes in peripheral immunological parameters were correlated with patients' outcome. In detail, 34 NSCLC patients were enrolled and classified as progressors (P) or non-progressors (NP), according to the RECIST evaluation. We demonstrated a reduction in pro-inflammatory cytokines such as IL-8, IL-6, and IL-1ß in only NP patients after ICI treatment. Moreover, using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis, we characterized in NP patients a significant increase in the amount of lymphocytes and a slight contraction of myeloid cells such as neutrophils and monocytes. Despite this moderate ICI-associated alteration in myeloid cells, we identified a distinctive reduction of c-FLIP expression in M-MDSCs from NP patients concurrently with the first clinical evaluation (T1), even though NP and P patients showed the same level of expression at baseline (T0). In agreement with the c-FLIP expression, monocytes isolated from both P and NP patients displayed similar immunosuppressive functions at T0; however, this pro-tumor activity was negatively influenced at T1 in the NP patient cohort exclusively. Hence, ICI therapy can mitigate systemic inflammation and impair MDSC-dependent immunosuppression.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Células Supresoras de Origen Mieloide , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Monocitos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Background & Aims: Type 1 autoimmune pancreatitis (AIP) is an IgG4-related disease whose diagnosis is challenging. The aim of this study was to investigate the diagnostic value of circulating total and IgG4+ plasmablasts in differentiating this condition from the other main pancreatic diseases. Methods: Patients with type 1 AIP (n = 19) were prospectively enrolled in a tertiary center together with patients suffering from type 2 or not otherwise specified (NOS) AIP (n = 10), pancreatic adenocarcinoma (n = 17), chronic pancreatitis (n = 20), and intraductal papillary mucinous neoplasia or chronic asymptomatic pancreatic hyperenzymemia (n = 21) as control groups. Flow cytometry was used to measure the total plasmablast and IgG4+ plasmablast number by gating peripheral blood CD45+CD19+CD38hiCD20-CD24-CD27+ and CD45+CD19+CD38hiCD20-CD24-CD27+IgG4+ cells, respectively. In patients with AIP, these cell populations were also evaluated after 1 month of therapy, after 2-4 months from the end of treatment, and after 1 year from the enrollment. The study was approved by the local ethics committee (protocol number: 59133, 30/11/2017). Results: Total plasmablast quantification was capable of discriminating type 1 AIP from all the other pancreatic disorders with a sensitivity of 47% and a specificity of 81%, according to a cutoff of 4500 cells/mL (AUC = 0.738), whereas IgG4+ plasmablast count distinguished type 1 AIP from all the other pancreatic disorders with a sensitivity of 80% and a specificity of 97% when applying a cutoff of 210 IgG4+ cells/mL (AUC = 0.879). The basal IgG4+ plasmablast number was significantly higher (P = .0001) in type 1 AIP than in type 2/NOS AIP, decreased after steroid therapy, and increased at disease relapse. Conclusion: IgG4+ plasmablast count represents a potentially useful biomarker to differentiate type 1 from type 2/NOS AIP and from other pancreatic diseases.
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Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-CoV-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homolog in myeloid cells triggered a STAT3-linked, progressive, and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury, and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state.
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COVID-19/fisiopatología , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/metabolismo , Inflamación/metabolismo , Factor de Transcripción STAT3/metabolismo , Anciano , Anciano de 80 o más Años , Animales , COVID-19/metabolismo , Caspasa 8/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , SARS-CoV-2/inmunología , Factor de Transcripción STAT3/genética , Transducción de SeñalRESUMEN
Alexander's disease (AxD) is a rare, usually relentlessly progressive disorder of astroglial cells in the central nervous system related to mutations in the gene encoding the type III intermediate filament protein, glial fibrillary acidic protein (GFAP). The pathophysiology of AxD is only partially understood. Available data indicate that an excessive GFAP gene expression may play a role. In particular, a "threshold hypothesis" has been reported, suggesting that mutant GFAP representing about 20% of the total cellular GFAP should be sufficient to cause disease. Thus, strategies based on reducing cellular mutant GFAP protein levels and/or activating biological processes involved in the correct protein folding could be effective in counteracting the toxic effect of misfolded GFAP. Considering that clomipramine (CLM), which has been selected by a wide small molecules screening as the greatest inhibitory potential drug against GFAP expression, is contraindicated because of its proconvulsant activity in the infantile form of AxD, which is also characterized by the occurrence of epileptic seizures, two powerful antiepileptic agents, carbamazepine (CBZ) and phenytoin (PHT), which share specific stereochemical features in common with CLM, were taken into consideration in a reliable in vitro model of AxD. In the present work, we document for the first time that CBZ and PHT have a definite inhibitory effect on pathological GFAP cellular expression and folding. Moreover, we confirm previous results of a similar beneficial effect of CLM. In addition, we have demonstrated that CBZ and CLM play a refolding effect on mutant GFAP proteins, likely ascribed at the induction of CRYAB expression, resulting in the decrease of mutant GFAP aggregates formation. As CBZ and PHT are currently approved for use in humans, their documented effects on pathological GFAP cellular expression and folding may indicate a potential therapeutic role as disease-modifying agents of these drugs in the clinical management of AxD, particularly in AxD patients with focal epilepsy with and without secondary generalization.
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Expression of the L-arginine catabolizing enzyme arginase 1 (ARG1) is a central immunosuppressive mechanism mediated by tumor-educated myeloid cells. Increased activity of ARG1 promotes the formation of an immunosuppressive microenvironment and leads to a more aggressive phenotype in many cancers. Intrinsic T-cell immunity against ARG1-derived epitopes in the peripheral blood of cancer patients and healthy subjects has previously been demonstrated. To evaluate the antitumor efficacy of ARG1-derived peptide vaccines as a monotherapy and as a combinational therapy with checkpoint blockade, different in vivo syngeneic mouse tumor models were utilized. To evaluate the antitumor effects, flow cytometry analysis and IHC were performed on tumors, and ELISPOT assays were performed to characterize immune responses. We show that ARG1-targeting therapeutic vaccines were able to activate endogenous antitumor immunity in several in vivo syngeneic mouse tumor models and to modulate the cell composition of the tumor microenvironment without causing any associated side effects or systemic toxicity. ARG1-targeting vaccines in combination with anti-PD-1 also resulted in increased T-cell infiltration, decreased ARG1 expression, reduced suppressive function of tumor-educated myeloid cells, and a shift in the M1/M2 ratio of tumor-infiltrating macrophages. These results indicated that the induced shift toward a more proinflammatory microenvironment by ARG1-targeting immunotherapy favors effective tumor control when combined with anti-PD-1 checkpoint blockade. Our data illustrate the ability of ARG1-based immune modulatory vaccination to elicit antigen-specific immunosurveillance and imply the feasibility of this novel immunotherapeutic approach for clinical translation.
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Arginasa/metabolismo , Células Mieloides/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Vacunas/uso terapéutico , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Microambiente Tumoral , Vacunas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Plasticity and adaptation to environmental stress are the main features that tumor and immune system share. Except for intrinsic and high-defined properties, cancer and immune cells need to overcome the opponent's defenses by activating more effective signaling networks, based on common elements such as transcriptional factors, protein-based complexes and receptors. Interestingly, growing evidence point to an increasing number of proteins capable of performing diverse and unpredictable functions. These multifunctional proteins are defined as moonlighting proteins. During cancer progression, several moonlighting proteins are involved in promoting an immunosuppressive microenvironment by reprogramming immune cells to support tumor growth and metastatic spread. Conversely, other moonlighting proteins support tumor antigen presentation and lymphocytes activation, leading to several anti-cancer immunological responses. In this light, moonlighting proteins could be used as promising new potential targets for improving current cancer therapies. In this review, we describe in details 12 unprecedented moonlighting proteins that during cancer progression play a decisive role in guiding cancer-associated immunomodulation by shaping innate or adaptive immune response.
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Neoplasias/inmunología , Proteínas/inmunología , Animales , Proliferación Celular/fisiología , Humanos , Inmunidad/inmunología , Inmunomodulación/inmunología , Activación de Linfocitos/inmunologíaRESUMEN
Extracellular vesicles (EVs) are membrane-coated particles secreted by virtually all cell types in response to different stimuli, both in physiological and pathological conditions. Their content generally reflects their biological functions and includes a variety of molecules, such as nucleic acids, proteins and cellular components. The role of EVs as signaling vehicles has been widely demonstrated. In particular, they are actively involved in the pathogenesis of several hematological malignancies (HM), mainly interacting with a number of target cells and inducing functional and epigenetic changes. In this regard, by releasing their cargo, EVs play a pivotal role in the bilateral cross-talk between tumor microenvironment and cancer cells, thus facilitating mechanisms of immune escape and supporting tumor growth and progression. Recent advances in high-throughput technologies have allowed the deep characterization and functional interpretation of EV content. In this review, the current knowledge on the high-throughput technology-based characterization of EV cargo in HM is summarized.
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In this study, we compared the overall gene and pathway expression profiles of HS-5 and HS-27A stromal cell lines with those of primary bone marrow MSCs to verify if they can be considered a reliable alternative tool for evaluating the contribution of MSCs in tumor development and immunomodulation. Indeed, due to their easier manipulation in vitro as compared to primary MSC cultures, several published studies took advantage of stromal cell lines to assess the biological mechanisms mediated by stromal cells in influencing tumor biology and immune responses. However, the process carried out to obtain immortalized cell lines could profoundly alter gene expression profile, and consequently their biological characteristics, leading to debatable results. Here, we evaluated the still undisclosed similarities and differences between HS-5, HS-27A cell lines and primary bone marrow MSCs in the context of tumor development and immunomodulation. Furthermore, we assessed by standardized immunological assays the capability of the cell lines to reproduce the general mechanisms of MSC immunoregulation. We found that only HS-5 cell line could be suitable to reproduce not only the MSC capacity to influence tumor biology, but also to evaluate the molecular mechanisms underlying tumor immune escape mediated by stroma cells. However, HS-5 pre-treatment with inflammatory cytokines, that normally enhances the immunosuppressive activity of primary MSCs, did not reproduce the same MSCs behavior, highlighting the necessity to accurately set up in vitro assays when HS-5 cell line is used instead of its primary counterpart.
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Wnt/ß-catenin signaling has been reported in Acute Myeloid leukemia, but little is known about its significance as a prognostic biomarker and drug target. In this study, we first evaluated the correlation between expression levels of Wnt molecules and clinical outcome. Then, we studied-in vitro and in vivo-the anti-leukemic value of combinatorial treatment between Wnt inhibitors and classic anti-leukemia drugs. Higher levels of ß-catenin, Ser675-phospho-ß-catenin and GSK-3α (total and Ser 9) were found in AML cells from intermediate or poor risk patients; nevertheless, patients presenting high activity of Wnt/ß-catenin displayed shorter progression-free survival (PFS) according to univariate analysis. In vitro, many pharmacological inhibitors of Wnt signalling, i.e., LRP6 (Niclosamide), GSK-3 (LiCl, AR-A014418), and TCF/LEF (PNU-74654) but not Porcupine (IWP-2), significantly reduced proliferation and improved the drug sensitivity of AML cells cultured alone or in the presence of bone marrow stromal cells. In vivo, PNU-74654, Niclosamide and LiCl administration significantly reduced the bone marrow leukemic burden acting synergistically with Ara-C, thus improving mouse survival. Overall, our study demonstrates the antileukemic role of Wnt/ß-catenin inhibition that may represent a potential new therapeutics strategy in AML.
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In this study, a successful medicinal chemistry campaign that exploited virtual, biophysical, and biological investigations led to the identification of a novel class of IDO1 inhibitors based on a benzimidazole substructure. This family of compounds is endowed with an extensive bonding network in the protein active site, including the interaction with pocket C, a region not commonly exploited by previously reported IDO1 inhibitors. The tight packing of selected compounds within the enzyme contributes to the strong binding interaction with IDO1, to the inhibitory potency at the low nanomolar level in several tumoral settings, and to the selectivity toward IDO1 over TDO and CYPs. Notably, a significant reduction of L-Kyn levels in plasma, together with a potent effect on abrogating immunosuppressive properties of MDSC-like cells isolated from patients affected by pancreatic ductal adenocarcinoma, was observed, pointing to this class of molecules as a valuable template for boosting the antitumor immune system.