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Marine invertebrates are a traditional source of natural products with relevant biological properties. Tunicates are soft-bodied, solitary or colonial, sessile organisms that provide compounds unique in their structure and activity. The aim of this work was to investigate the chemical composition of the ascidian Cystodytes dellechiajei, selected on the basis of a positive result in biological screening for ligands of relevant receptors of the innate immune system, including TLR2, TLR4, dectin-1b, and TREM2. Bioassay-guided screening of this tunicate extract yielded two known pyridoacridine alkaloids, shermilamine B (1) and N-deacetylshermilamine B (2), and a family of methyl-branched cerebrosides (3). Compounds 2 and 3 showed selective binding to TREM2 in a dose-dependent manner. N-deacetylshermilamine B (2), together with its acetylated analogue, shermilamine B (1), was also strongly cytotoxic against multiple myeloma cell lines. TREM2 is involved in immunomodulatory processes and neurodegenerative diseases. N-deacetylshermilamine B (2) is the first example of a polycyclic alkaloid to show an affinity for this receptor.
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Alcaloides , Antineoplásicos , Urocordados , Animales , Urocordados/química , Alcaloides/farmacología , Alcaloides/química , Antineoplásicos/farmacología , Antineoplásicos/químicaRESUMEN
Immunogenic Cell Death (ICD) represents a mechanism of enhancing T cell-driven response against tumor cells. The process is enabled by release of damage-associated molecular patterns (DAMPs) and cytokines by dying cells. Based on molecular studies and clinical marker assessment, ICD can be a new target for cancer chemotherapy hitherto restricted to a few conventional anticancer drugs. In view of the development of small molecules in targeted cancer therapy, we reported the preliminary evidence on the role of the natural product lepadin A (1) as a novel ICD inducer. Here we describe the ICD mechanism of lepadin A (1) by proving the translocation of the protein calreticulin (CRT) to the plasma membrane of human A2058 melanoma cells. CRT exposure is an ICD marker in clinical studies and was associated with the activation of the intrinsic apoptotic pathway in A2058 cells with lepadin A (1). After the treatment, the tumour cells acquired the ability to activate dendritic cells (DCs) with cytokine release and costimulatory molecule expression that is consistent with a phenotypic profile committed to priming T lymphocytes via a CD91-dependent mechanism. The effect of lepadin A (1) was dose-dependent and comparable to the response of the chemotherapy drug doxorubicin (2), a well-established ICD inducer.
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Introduction: Sulfavant A (SULF A) is a synthetic derivative of naturally occurring sulfolipids. The molecule triggers TREM2-related maturation of dendritic cells (DCs) and has shown promising adjuvant activity in a cancer vaccine model. Methods: the immunomodulatory activity of SULF A is tested in an allogeneic mixed lymphocyte reaction (MLR) assay based on monocyte-derived dendritic cells and naïve T lymphocytes from human donors. Flow cytometry multiparametric analyses and ELISA assays were performed to characterize the immune populations, T cell proliferation, and to quantify key cytokines. Results: Supplementation of 10 µg/mL SULF A to the co-cultures induced DCs to expose the costimulatory molecules ICOSL and OX40L and to reduce release of the pro-inflammatory cytokine IL-12. After 7 days of SULF A treatment, T lymphocytes proliferated more and showed increased IL-4 synthesis along with downregulation of Th1 signals such as IFNγ, T-bet and CXCR3. Consistent with these findings, naïve T cells polarized toward a regulatory phenotype with up-regulation of FOXP3 expression and IL-10 synthesis. Flow cytometry analysis also supported the priming of a CD127-/CD4+/CD25+ subpopulation positive for ICOS, the inhibitory molecule CTLA-4, and the activation marker CD69. Discussion: These results prove that SULF A can modulate DC-T cell synapse and stimulate lymphocyte proliferation and activation. In the hyperresponsive and uncontrolled context of the allogeneic MLR, the effect is associated to differentiation of regulatory T cell subsets and dampening of inflammatory signals.
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Adyuvantes Inmunológicos , Trasplante de Células Madre Hematopoyéticas , Glicoproteínas de Membrana , Receptores Inmunológicos , Humanos , Adyuvantes Inmunológicos/farmacología , Interleucina-12 , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Glicoproteínas de Membrana/agonistas , Receptores Inmunológicos/agonistasRESUMEN
OBJECTIVE: The immune response arises from a fine balance of mechanisms that provide for surveillance, tolerance, and elimination of dangers. Sulfavant A (SULF A) is a sulfolipid with a promising adjuvant activity. Here we studied the mechanism of action of SULF A and addressed the identification of its molecular target in human dendritic cells (hDCs). METHODS: Adjuvant effect and immunological response to SULF A were assessed on DCs derived from human donors. In addition to testing various reporter cells, target identification and downstream signalling was supported by a reverse pharmacology approach based on antibody blocking and gene silencing, crosstalk with TLR pathways, use of human allogeneic mixed lymphocyte reaction. RESULTS: SULF A binds to the Triggering Receptor Expressed on Myeloid cells-2 (TREM2) and initiates an unconventional maturation of hDCs leading to enhanced migration activity and up-regulation of MHC and co-stimulatory molecules without release of conventional cytokines. This response involves the SYK-NFAT axis and is compromised by blockade or gene silencing of TREM2. Activation by SULF A preserved the DC functions to excite the allogeneic T cell response, and increased interleukin-10 release after lipopolysaccharide stimulation. CONCLUSION: SULF A is the first synthetic small molecule that binds to TREM2. The receptor engagement drives differentiation of an unprecedented DC phenotype (homeDCs) that contributes to immune homeostasis without compromising lymphocyte activation and immunogenic response. This mechanism fully supports the adjuvant and immunoregulatory activity of SULF A. We also propose that the biological properties of SULF A can be of interest in various physiopathological mechanisms and therapies involving TREM2.
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Células Dendríticas , Activación de Linfocitos , Citocinas/metabolismo , Células Dendríticas/metabolismo , Homeostasis , LigandosRESUMEN
Natural products and their synthetic analogs and derivatives are a traditional source of bioactive molecules with potential development as drug candidates. In this context, Marine Natural Products (MNPs) represent a rich reservoir of diverse molecular skeletons with potential pharmacological activity that, so far, has been mostly explored in cancer and infectious diseases. Starting from the development of a novel bioassay-guided screening platform for immunomodulatory compounds from an in-house MNPs library, we report the identification of the alkaloid lepadin A as a new model compound for immune-based anticancer activity with characteristics that suggest a possible mechanism as Immunogenic Cell Death inducer. The work describes the molecular-based bioprospecting in the Gulf of Naples together with the bioassay-guided fractionation, the chemical characterization of the alkaloid, and the biological activity in mouse dendritic cells (D1).
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Alcaloides , Antineoplásicos , Productos Biológicos , Alcaloides/química , Alcaloides/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Productos Biológicos/química , Muerte Celular Inmunogénica , Ratones , QuinolinasRESUMEN
Several studies have shown that T-cells might be involved in pathophysiology of acute coronary syndromes (ACS). Tissue factor (TF) plays a key role in ACS. Many evidences have indicated that some statins reduce TF expression in several cell types. However, literature about rosuvastatin and TF and about statins effects on T-cells is still scanty. Colchicine is an anti-inflammatory drug recently proven to have beneficial effects in ACS via unknown mechanisms. This study investigates the effects of colchicine and rosuvastatin on TF expression in oxLDL-activated T-cells. T-cells, isolated from buffy coats of healthy volunteers, were stimulated with oxLDL (50 µg/dL). T-cells were pre-incubated with colchicine (10 µM) or rosuvastatin (5 µM) for 1 h and then stimulated with oxLDL (50 µg/mL). TF gene (RT-PCR), protein (western blot), surface expression (FACS) and procoagulant activity (FXa generation assay) were measured. NF-κB/IκB axis was examined by western blot analysis and translocation assay. Colchicine and rosuvastatin significantly reduced TF gene, and protein expression and procoagulant activity in oxLDL stimulated T-cells. This effect was associated with a significant reduction in TF surface expression as well as its procoagulant activity. These phenomena appear modulated by drug effects on the transcription factor NF-kB. Rosuvastatin and colchicine prevent TF expression in oxLDL-stimulated T-cells by modulating the NF-κB/IκB axis. Thus, we speculate that this might be another mechanism by which these drugs exert benefic cardiovascular effects.
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Síndrome Coronario Agudo , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Síndrome Coronario Agudo/tratamiento farmacológico , Colchicina/farmacología , Humanos , Lipoproteínas LDL , FN-kappa B/metabolismo , Rosuvastatina Cálcica/farmacología , Linfocitos T/metabolismo , Tromboplastina/genéticaRESUMEN
The marine environment is potentially a prolific source of small molecules with significant biological activities. In recent years, the development of new chromatographic phases and the progress in cell and molecular techniques have facilitated the search for marine natural products (MNPs) as novel pharmacophores and enhanced the success rate in the selection of new potential drug candidates. However, most of this exploration has so far been driven by anticancer research and has been limited to a reduced number of taxonomic groups. In this article, we report a test study on the screening potential of an in-house library of natural small molecules composed of 285 samples derived from 57 marine organisms that were chosen from among the major eukaryotic phyla so far represented in studies on bioactive MNPs. Both the extracts and SPE fractions of these organisms were simultaneously submitted to three different bioassays-two phenotypic and one enzymatic-for cytotoxic, antidiabetic, and antibacterial activity. On the whole, the screening of the MNP library selected 11 potential hits, but the distribution of the biological results showed that SPE fractionation increased the positive score regardless of the taxonomic group. In many cases, activity could be detected only in the enriched fractions after the elimination of the bulky effect due to salts. On a statistical basis, sponges and molluscs were confirmed to be the most significant source of cytotoxic and antimicrobial products, but other phyla were found to be effective with the other therapeutic targets.
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Antineoplásicos/farmacología , Organismos Acuáticos , Animales , Antineoplásicos/química , Fraccionamiento Químico , Descubrimiento de Drogas , Moluscos , PoríferosRESUMEN
A subset of colorectal cancer (CRC) with a mesenchymal phenotype (CMS4) displays an aggressive disease, with an increased risk of recurrence after surgery, reduced survival, and resistance to standard treatments. It has been shown that the AXL and TGFß signaling pathways are involved in epithelial-to-mesenchymal transition, migration, metastatic spread, and unresponsiveness to targeted therapies. However, the prognostic role of the combination of these biomarkers and the anti-tumor effect of AXL and TGFß inhibition in CRC still has to be assessed. To evaluate the role of AXL and TGFß as negative biomarker in CRC, we conducted an in-depth in silico analysis of CRC samples derived from the Gene Expression Omnibus. We found that AXL and TGFß receptors are upregulated in CMS4 tumors and are correlated with an increased risk of recurrence after surgery in stage II/III CRC and a reduced overall survival. Moreover, we showed that AXL receptor is differently expressed in human CRC cell lines. Dual treatment with the TGFß galunisertib and the AXL inhibitor, bemcentinib, significantly reduced colony formation and migration capabilities of tumor cells and displayed a strong anti-tumor activity in 3D spheroid cultures derived from patients with advanced CRC. Our work shows that AXL and TGFß receptors identify a subgroup of CRC with a mesenchymal phenotype and correlate with poor prognosis. Dual inhibition of AXL and TGFß could represent a novel therapeutic strategy for patients with this aggressive disease.
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Adenocarcinoma/tratamiento farmacológico , Benzocicloheptenos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Transición Epitelial-Mesenquimal , Recurrencia Local de Neoplasia/tratamiento farmacológico , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirazoles/farmacología , Quinolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor Tipo II de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Triazoles/farmacología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Línea Celular Tumoral , Movimiento Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Biología Computacional/métodos , Bases de Datos Genéticas , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal , Esferoides Celulares , Tirosina Quinasa del Receptor AxlRESUMEN
Immunotherapy takes advantage of the immune system to prevent, control, and eliminate neoplastic cells. The research in the field has already led to major breakthroughs to treat cancer. In this work, we describe a platform that integrates in vitro bioassays to test the immune response and direct antitumor effects for the preclinical discovery of anticancer candidates. The platform relies on the use of dendritic cells that are professional antigen-presenting cells (APC) able to activate T cells and trigger a primary adaptive immune response. The experimental procedure is based on two phenotypic assays for the selection of chemical leads by both a panel of nine tumor cell lines and growth factor-dependent immature mouse dendritic cells (D1). The positive hits are then validated by a secondary test on human monocyte-derived dendritic cells (MoDCs). The aim of this approach is the selection of potential immunotherapeutic small molecules from natural extracts or chemical libraries.
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Antineoplásicos/farmacología , Bioensayo , Células Dendríticas/efectos de los fármacos , Descubrimiento de Drogas , Inmunoterapia , Neoplasias/tratamiento farmacológico , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Inmunidad Innata/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Fenotipo , Bibliotecas de Moléculas Pequeñas , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Anti-PD-1 antibodies revolutionized the treatment of advanced melanoma patients. However, one out of three do not respond to this therapy, with an overall poor prognosis. Identification of predictive biomarkers in patients receiving immune-based therapies is necessary for minimizing risk of toxicity and optimizing patient benefit and is still an important unmet clinical need. Recently, many studies have evaluated peripheral blood markers as potential biomarkers, but none so far have been validated. We collected at baseline peripheral blood samples from 18 consecutive advanced melanoma patients treated with anti-PD-1 therapy. Main pro- and anti-inflammatory cytokines were studied in PBMCs from baseline blood samples both evaluating mRNA expression by qRT-PCR and identifying PBMCs subpopulations by FACS analysis. We found that IFN-γ mRNA expression levels were significantly higher in responder patients compared to non-responder ones. Moreover, to better validate its role, we evaluated the IFN-γ/IL-10 ratio. This value was higher in responder patients. FACS analysis confirmed that CD4 + IFN-γ + PBMCs percentage was higher in responders. Our data suggest an interesting correlation between IFN-γ/IL-10 ratio and response to anti-PD-1 therapy in advanced melanoma patients, suggesting a new biomarker that could be easily incorporated in clinical practice.
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Antineoplásicos Inmunológicos/uso terapéutico , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Leucocitos Mononucleares/metabolismo , Melanoma/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Melanoma/metabolismo , Persona de Mediana Edad , Pronóstico , Neoplasias Cutáneas/metabolismoRESUMEN
BACKGROUND: Antibody-dependent cell-mediated cytotoxicity (ADCC) may mediate antitumour activity of IgG1-isotype monoclonal antibody (mAb), suggesting as potential treatment combination of IgG1-mAbs, anti-epidermal growth factor receptor cetuximab and anti-programmed death-ligand-1 avelumab. METHODS: We evaluated ADCC induction in lung cancer cells by lactate dehydrogenase (LDH) release assay. Antitumour activity and safety of cetuximab plus avelumab were explored in a single-arm proof-of-concept study in pre-treated non-small cell lung cancer (NSCLC) patients (pt) (Cetuximab-AVElumab-lung, CAVE-Lung). Search for predictive biomarkers of response was done. RESULTS: Avelumab plus cetuximab induced ADCC in NSCLC cells in vitro in presence of natural killers (NK) from healthy donors (HD) or NSCLC pt, as effectors. Sixteen relapsed NSCLC pt were treated with avelumab plus cetuximab. Antitumour activity was observed in 6/16 pt, defined by progression free survival (PFS) ≥8 months, with 4 of them still on treatment at data lock time (range, 14-19 months). Of note, 3/6 responders had received as previous line anti-programmed death-1 therapy. In responders, clinical benefit was accompanied by significant increase in LDH release over baseline at the first radiological evaluation (8 weeks) (p=0.01) and by early skin toxicity; while in the 10 non-responders, that had PFS ≤5 months, LDH release tends to reduce. Baseline circulating DNA levels were higher in non-responders compared with responders and HD (p=0.026) and decrease in responders during therapy. Mutations in DNA damage responsive family genes were found in responders. CONCLUSION: Cetuximab and avelumab activates NSCLC pt NK cells. Ex vivo evaluation of ADCC, circulating DNA levels and early skin toxicity may predict response to cetuximab plus avelumab in NSCLC.EUDRACT 2017-004195-58.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Cetuximab/farmacología , Cetuximab/uso terapéutico , Humanos , Inmunoglobulina G , Células Asesinas Naturales , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genéticaRESUMEN
Tissue-resident memory (Trm) cells are specialized components of both CD4+ and CD8+ T cell subsets that persist in peripheral nonlymphoid tissues following infections and provide fast response in case of a secondary invasion by the same pathogen. Trm cells express the surface markers CD69, CD103, and the immune checkpoint molecule PD-1. Trm cells develop not only in the context of infections but also in tumors, where they can provide a line of defense as suggested by the positive correlation between the frequency of tumor-infiltrating Trm cells and patients' survival. Trm cells persistence in peripheral tissues depends on their adaptation to the local microenvironment and the presence of survival factors, mainly IL-7, IL-15, and Notch ligands. However, the cell sources of these factors are largely unknown, especially in the context of tumors. Here, we show that head-neck squamous cell carcinoma (HNSCC) is enriched in CD4+ and CD8+ T cells with a Trm phenotype. Moreover, we show that mesenchymal stromal cells that accumulate in HNSCC are a source of survival factors and allow proper expression of Trm-typical markers in a VCAM1-dependent manner.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Escamosas/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Células Madre Mesenquimatosas/inmunología , Comunicación Celular , Movimiento Celular , Células Cultivadas , Humanos , Memoria Inmunológica , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Fenotipo , Receptores Notch/metabolismo , Microambiente Tumoral , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Background: Systemic sclerosis (SSc) T cells can induce apoptosis of autologous skin fibroblasts in vitro. Th17 cells have been reported to increase in SSc patients, and interleukin-17A (IL-17A) has a profibrotic function. We used a system based on T-cell-autologous fibroblast co-cultures to further investigate a possible role of IL-17A in SSc. Methods: T cells from diffuse SSc patients were co-cultured with autologous skin fibroblasts. IL17A mRNA was assessed by real-time PCR in co-cultured and control T cells, while IL17RA, CXCL1, CCL2, CCL3, COL1A1, COL3A1, CTGF, TGFBR2, and SMAD3 mRNAs were assessed in co-cultured and control fibroblasts. In subset experiments, co-cultures and control cells were treated with either IL-17A or IL-17A plus anti-IL17 receptor monoclonal antibody (α-IL-17RA mAb). Chemokine and procollagen type I (PCI) production was further investigated at the protein level in cell culture supernatants by multiple suspension immunoassay and sandwich ELISA, respectively. Co-cultured and control fibroblasts were also stained with Annexin V and analyzed by flow cytometry. Results: T cell-fibroblast co-cultures overexpressed IL17A and IL17RA. Furthermore, co-cultured fibroblasts upregulated IL-17A targets CXCL1, CCL2, and CCL3, while COL1A1, COL3A1, CTGF, and two key effectors of the TGF-ß signaling, TGFBR2 and SMAD3, were found downregulated. Consistently, chemokine concentrations were increased in co-culture supernatants, while PCI levels were reduced, especially after stimulation with ectopic IL-17A. Finally, simultaneous α-IL-17RA mAb treatment restored PCI levels and reduced fibroblast apoptosis in IL-17A-stimulated co-cultures. Conclusion: These data suggest that IL-17A upregulation might play a role in modulating T cell-mediated antifibrotic and proapoptotic effects in co-cultured autologous skin fibroblasts.
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Fibroblastos/metabolismo , Interleucina-17/metabolismo , Esclerodermia Sistémica/inmunología , Piel/patología , Células Th17/inmunología , Adulto , Anticuerpos Bloqueadores/metabolismo , Apoptosis , Autoantígenos/inmunología , Células Cultivadas , Colágeno Tipo I/metabolismo , Femenino , Fibroblastos/patología , Fibrosis , Humanos , Masculino , Persona de Mediana Edad , Receptores de Interleucina-17/inmunología , Transducción de Señal , Regulación hacia Arriba , Adulto JovenRESUMEN
AIMS: T-lymphocytes plays an important role in the pathophysiology of acute coronary syndromes. T-cell activation in vitro by pro-inflammatory cytokines may lead to functional tissue factor (TF) expression, indicating a possible contribution of immunity to thrombosis. Oxidized low-density lipoproteins (oxLDLs) are found abundantly in atherosclerotic plaques. We aimed at evaluating the effects of oxLDLs on TF expression in T cells and the role of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). METHODS AND RESULTS: CD3+ cells were isolated from healthy volunteers. Gene, protein, and surface expression of TF, as well as of LOX-1, were assessed at different time-points after oxLDL stimulation. To determine whether oxLDL-induced TF was LOX-1 dependent, T cells were pre-incubated with an LOX-1 inhibiting peptide (L-RBP) or with an anti-LOX-1 blocking antibody. To exclude that TF expression was mediated by reactive oxygen species (ROS) generation, oxLDL-stimulated T cells were pre-incubated with superoxide dismutase + catalase or with 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), an intracellular free radical scavenger. Finally, to determine if the observed findings in vitro may have a biological relevance, the presence of CD3+/TF+/LOX-1+ cells was evaluated by immunofluorescence in human carotid atherosclerotic lesions. oxLDLs induced functionally active TF expression in T cells in a dose- and time-dependent manner, independently on ROS generation. No effect was observed in native LDL-treated T cells. LOX-1 expression was also induced by oxLDLs in a time- and dose-dependent manner. Pre-incubation with L-RBP or anti-LOX-1 antibody almost completely inhibited oxLDL-mediated TF expression. Interestingly, human carotid plaques showed significant infiltration of CD3+ cells (mainly CD8+ cells), some of which were positive for both TF and LOX-1. CONCLUSION: oxLDLs induce functional TF expression in T-lymphocytes in vitro via interaction of oxLDLs with LOX-1. Human carotid atherosclerotic plaques contain CD3+/CD8+cells that express both TF and LOX-1, indicating that also in patients these mechanisms may play an important role.
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Enfermedades de las Arterias Carótidas/metabolismo , Lipoproteínas LDL/farmacología , Receptores Depuradores de Clase E/agonistas , Linfocitos T/efectos de los fármacos , Tromboplastina/metabolismo , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Células Cultivadas , Humanos , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Placa Aterosclerótica , Especies Reactivas de Oxígeno/metabolismo , Receptores Depuradores de Clase E/genética , Receptores Depuradores de Clase E/metabolismo , Linfocitos T/metabolismo , Tromboplastina/genética , Regulación hacia ArribaRESUMEN
Manipulation of the immune response is a game changer in lung cancer treatment, revolutionizing management. PD1 and CTLA4 are dynamically expressed on different T cell subsets that can either disrupt or sustain tumor growth. Monoclonal antibodies (MoAbs) against PD1/PDL1 and CTLA4 have shown that inhibitory signals can be impaired, blocking T cell activation and function. MoAbs, used as both single-agents or in combination with standard therapy for the treatment of advanced non-small cell lung cancer (NSCLC), have exhibited advantages in terms of overall survival and response rate; nivolumab, pembrolizumab, atezolizumab and more recently, durvalumab, have already been approved for lung cancer treatment and more compounds are in the pipeline. A better understanding of signaling elicited by these antibodies on T cell subsets, as well as identification of biological determinants of sensitivity, resistance and correlates of efficacy, will help to define the mechanisms of antitumor responses. In addition, the relevance of T regulatory cells (Treg) involved in immune responses in cancer is attracting increasing interest. A major challenge for future research is to understand why a durable response to immune checkpoint inhibitors (ICIs) occurs only in subsets of patients and the mechanisms of resistance after an initial response. This review will explore current understanding and future direction of research on ICI treatment in lung cancer and the impact of tumor immune microenvironment n influencing clinical responses.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor/análisis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Antineoplásicos Inmunológicos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Receptores Coestimuladores e Inhibidores de Linfocitos T/inmunología , Humanos , Neoplasias Pulmonares/inmunologíaRESUMEN
INTRODUCTION: In the era of precision medicine, research studies are aiming to design patient-tailored treatment strategies. In this work, we present a clinical case of a patient with non-small cell lung cancer (NSCLC) accompanied by a translational study with the intent to assess the correspondence of drug sensitivity in ex vivo spheroidal tumour cultures and peripheral blood biomarkers with clinical outcome. METHODS: Primary tumour tissue, patient-derived tumour spheroids, peripheral blood mononuclear cells and circulating DNA were analysed to assess drug sensitivity and immunological profiling, and all these data were correlated with clinical and radiological evaluations. RESULTS: Immunohistochemistry, immunofluorescence, next generation sequencing analysis and T-lymphocyte receptor repertoire assay results showed elevated concordance among primary tumour tissue, ex vivo three-dimensional tumour spheroid specimen and circulating DNA. Cisplatin-based chemotherapy and anti-programmed death 1 drug sensitivity assessed in spheroidal cultures were strictly consistent with patient clinical response to adjuvant chemotherapy and first-line immune therapy. CONCLUSION: These results revealed that ex vivo drug sensitivity testing in three-dimensional spheroidal culture can reproduce clinical response to chemotherapy and immunotherapy, with the potential to use those culture models to predict patients' outcome from anticancer treatments and, therefore, the feasibility to select individualised therapy.
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[This corrects the article DOI: 10.1021/acsomega.8b03304.].
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Adjuvants are components of vaccine that enhance the specific immune response against co-inoculated antigens. Recently, we reported the characterization of a synthetic sulfolipid named Sulfavant A (1) as a promising candidate of a novel class of molecular adjuvants based on the sulfoquinovosyl-diacylglycerol skeleton. Here, we report an improved synthesis of the sulfolipid scaffold, as well as the preparation of two analogs named Sulfavant-S (2) and Sulfavant-R (3) with enhanced property to modulate master immune targets such as human dendritic cells (DCs). According to the present approach, synthesis of 1 is reduced from 14 to 11 steps with nearly triplication of the overall yield (11%). The new members 2 and 3 elicit DC maturation at a concentration of 10 nM, which is 1000 times more potent than the parent molecule 1. Analysis of dynamic light scattering indicates self-assembly of Sulfavants and formation of colloidal particles with a small hydrodynamic radius (50 nm) for the epimers 2 and 3 and a larger radius (150 nm) for 1. The colloidal aggregates are responsible for the bell-shaped dose-response curve of these products. We conclude that the particle size also affects the equilibrium with free monomers, thus determining the effective concentration of the sulfolipid molecule at the cellular targets and the different immunological efficacy of 1-3. Sulfavants (1-3) do not show in vitro cytotoxicity at concentrations 105 higher than the dose that triggers maximal immune response, thus predicting a low level of toxicological risk in their formulation in vaccines.
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BACKGROUND: Anti-PD-1/PD-L1 drugs are effective as monotherapy in a proportion of NSCLC patients and there is a strong rationale for combining them with targeted therapy. Inhibition of MAPK pathway may have pleiotropic effects on the microenvironment. This work investigates the efficacy of combining MEK and PD-L1 inhibition in pre-clinical and ex-vivo NSCLC models. METHODS: We studied the effects of MEK inhibitors (MEK-I) on PD-L1 and MCH-I protein expression and cytokine production in vitro in NSCLC cell lines and in PBMCs from healthy donors and NSCLC patients, the efficacy of combining MEK-I with anti-PD-L1 antibody in ex-vivo human spheroid cultures obtained from fresh biopsies from NSCLC patients in terms of cell growth arrest, cytokine production and T-cell activation by flow cytometry. RESULTS: MEK-I modulates in-vitro the immune micro-environment through a transcriptionally decrease of PD-L1 expression, enhance of MHC-I expression on tumor cells, increase of the production of several cytokines, like IFNγ, IL-6, IL-1ß and TNFα. These effects trigger a more permissive anti-tumor immune reaction, recruiting immune cells to the tumor sites. We confirmed these data on ex-vivo human spheroids, showing a synergism of MEK and PD-L1 inhibition as result of both direct cancer cell toxicity of MEK-I and its immune-stimulatory effect on cytokine secretion profile of cancer cells and PBMCs with the induction of the ones that sustain an immune-reactive and inflammatory micro-environment. CONCLUSIONS: Our work shows the biological rationale for combining immunotherapy with MEK-I in a reproducible ex-vivo 3D-culture model, useful to predict sensitivity of patients to such therapies.
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Acrilonitrilo/análogos & derivados , Compuestos de Anilina/farmacología , Antineoplásicos Inmunológicos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Acrilonitrilo/farmacología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Activación de Linfocitos/inmunología , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
BACKGROUND: Pioglitazone, a synthetic peroxisome proliferator activated receptor (PPAR-γ) ligand, is known as an antidiabetic drug included in the thiazolidinediones (TZDs) class. It regulates the lipid and glucose cell metabolism and recently a role in the inhibition of numerous cancer cell processes has been described. METHODS: In our work we investigate the anti-tumor effects of pioglitazone in in vitro models of non small cell lung cancer (NSCLC) and also, we generated ex-vivo three-dimensional (3D) cultures from human lung adenocarcinoma (ADK) as a model to test drug efficacy observed in vitro. The inhibitory effect of pioglitazone on cell proliferation, apoptosis and cell invasion in a panel of human NSCLC cell lines was evaluated by multiple assays. RESULTS: Pioglitazone reduced proliferative and invasive abilities with an IC50 ranging between 5 and 10 µM and induced apoptosis of NSCLC cells. mRNA microarray expression profiling showed a down regulation of MAPK, Myc and Ras genes after treatment with pioglitazone; altered gene expression was confirmed by protein analysis in a dose-related reduction of survivin and phosphorylated proteins levels of MAPK pathway. Interestingly mRNA microarray analysis showed also that pioglitazone affects TGFß pathway, which is important in the epithelial-to-mesenchimal transition (EMT) process, by down-regulating TGFßR1 and SMAD3 mRNA expression. In addition, extracellular acidification rate (ECAR) and a proportional reduction of markers of altered glucose metabolism in treated cells demonstrated also cell bioenergetics modulation by pioglitazone. CONCLUSIONS: Data indicate that PPAR-γ agonists represent an attractive treatment tool and by suppression of cell growth (in vitro and ex vivo models) and of invasion via blockade of MAPK cascade and TGFß/SMADs signaling, respectively, and its role in cancer bioenergetics and metabolism indicate that PPAR-γ agonists represent an attractive treatment tool for NSCLC.