RESUMO
Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.
Assuntos
DNA/imunologia , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/imunologia , Proteínas Quinases S6 Ribossômicas 90-kDa/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Citosol/imunologia , Citosol/metabolismo , Citosol/virologia , DNA/genética , DNA/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células HEK293 , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Humanos , Imunização/métodos , Immunoblotting , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Ovalbumina/genética , Ovalbumina/imunologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
Cisplatin is the most widely used chemotherapeutic agent, and resistance of neoplastic cells against this cytoxicant poses a major problem in clinical oncology. Here, we explored potential metabolic vulnerabilities of cisplatin-resistant non-small human cell lung cancer and ovarian cancer cell lines. Cisplatin-resistant clones were more sensitive to killing by nutrient deprivation in vitro and in vivo than their parental cisplatin-sensitive controls. The susceptibility of cisplatin-resistant cells to starvation could be explained by a particularly strong dependence on glutamine. Glutamine depletion was sufficient to restore cisplatin responses of initially cisplatin-resistant clones, and glutamine supplementation rescued cisplatin-resistant clones from starvation-induced death. Mass spectrometric metabolomics and specific interventions on glutamine metabolism revealed that, in cisplatin-resistant cells, glutamine is mostly required for nucleotide biosynthesis rather than for anaplerotic, bioenergetic or redox reactions. As a result, cisplatin-resistant cancers became exquisitely sensitive to treatment with antimetabolites that target nucleoside metabolism.
Assuntos
Antimetabólitos/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Glutamina/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Morte Celular , Linhagem Celular Tumoral , Metabolismo Energético , Feminino , Humanos , Espectrometria de Massas , Metaboloma , Modelos Biológicos , Nucleotídeos/biossínteseRESUMO
Regulation of alternative splicing is one of the most efficient mechanisms to enlarge the proteomic diversity in eukaryotic organisms. Many viruses hijack the splicing machinery following infection to accomplish their replication cycle. Regarding the HBV, numerous reports have described alternative splicing events of the long viral transcript (pregenomic RNA), which also acts as a template for viral genome replication. Alternative splicing of HBV pregenomic RNAs allows the synthesis of at least 20 spliced variants. In addition, almost all these spliced forms give rise to defective particles, detected in the blood of infected patients. HBV-spliced RNAs have long been unconsidered, probably due to their uneasy detection in comparison to unspliced forms as well as for their dispensable role during viral replication. However, recent data highlighted the relevance of these HBV-spliced variants through (1) the trans-regulation of the alternative splicing of viral transcripts along the course of liver disease; (2) the ability to generate defective particle formation, putative biomarker of the liver disease progression; (3) modulation of viral replication; and (4) their intrinsic propensity to encode for novel viral proteins involved in liver pathogenesis and immune response. Altogether, tricky regulation of HBV alternative splicing may contribute to modulate multiple viral and cellular processes all along the course of HBV-related liver disease.
Assuntos
Processamento Alternativo , Vírus da Hepatite B/genética , Splicing de RNA , Genoma Viral , Humanos , ProteômicaRESUMO
Oncolytic viruses (OVs) represent a new class of cancer immunotherapeutics. Administration of OVs to cancer-bearing hosts induces two distinct immunities: antiviral and antitumor. While antitumor immunity is beneficial, antiviral immune responses are often considered detrimental for the efficacy of OV-based therapy. The existing dogma postulates that anti-OV immune responses restrict viral replication and spread, and thus reduce direct OV-mediated killing of cancer cells. Accordingly, a myriad of therapeutic strategies aimed at mitigating anti-OV immune responses is presently being tested. Here, we advocate that OV-induced antiviral immune responses hold intrinsic anticancer benefits and are essential for establishing clinically desired antitumor immunity. Thus, to achieve the optimal efficacy of OV-based cancer immunotherapies, strategic management of anti-OV immune responses is of critical importance.
Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Animais , Humanos , Imunidade , Imunização , Neoplasias/imunologia , Neoplasias/virologia , Replicação ViralRESUMO
Cancer cells are subjected to constant selection by the immune system, meaning that tumors that become clinically manifest have managed to subvert or hide from immunosurveillance. Immune control can be facilitated by induction of autophagy, as well as by polyploidization of cancer cells. While autophagy causes the release of ATP, a chemotactic signal for myeloid cells, polyploidization can trigger endoplasmic reticulum stress with consequent exposure of the "eat-me" signal calreticulin on the cell surface, thereby facilitating the transfer of tumor antigens into dendritic cells. Hence, both autophagy and polyploidization cause the emission of adjuvant signals that ultimately elicit immune control by CD8+ T lymphocytes. We investigated the possibility that autophagy and polyploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. Mass spectrometry led to the identification of peptides that were presented on major histocompatibility complex (MHC) class I molecules in an autophagy-dependent fashion or that were specifically exposed on the surface of polyploid cells, yet lost upon passage of such cells through immunocompetent (but not immunodeficient) mice. However, the preferential recognition of autophagy-competent and polyploid cells by the innate and cellular immune systems did not correlate with the preferential recognition of such peptides in vivo. Moreover, vaccination with such peptides was unable to elicit tumor growth-inhibitory responses in vivo. We conclude that autophagy and polyploidy increase the immunogenicity of cancer cells mostly by affecting their adjuvanticity rather than their antigenicity.
Assuntos
Adjuvantes Imunológicos , Antígenos de Neoplasias/imunologia , Morte Celular , Vigilância Imunológica , Neoplasias/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Estresse do Retículo Endoplasmático , Humanos , Camundongos , Monitorização Imunológica , Transdução de SinaisRESUMO
PURPOSE OF REVIEW: Considering the failure of standard treatments (i.e. surgery, radiotherapy, chemotherapy) in treating cholangiocarcinoma (CCA), introduction of alternative interventions is urgently needed. During the past 2 decades, discoveries of the mechanisms of cancer immunosurveillance and tumor immune evasion have precipitated the emergence and clinical approval of immunotherapies in multiple malignant indications. Interest in their introduction for the care of CCA is recent and several immunotherapeutic approaches are undergoing a clinical evaluation. Undoubtedly, their efficient application, as monotherapy or in combination regimens, will rely on a deeper understanding of CCA immune contexture. RECENT FINDINGS: CCA cells appeared very potent in recruiting protumorigenic cells and shaping an immunosuppressive microenvironment. Elevated densities of several immune cells with immunoinhibitory activities within the malignant bed have been associated with poor prognosis in patients. Particularly, macrophages and neutrophils (especially in their alternatively activated phenotype) were pointed out for their role in cancer progression. Dendritic cells were described as ineffective in priming CCA-specific T-cell responses. SUMMARY: Quantitative and qualitative assessment of the innate and adaptive immune compartments of the CCA immune contexture, as well as their prognostic value, will benefit to the development of improved immunotherapeutic strategies.
Assuntos
Neoplasias dos Ductos Biliares/imunologia , Colangiocarcinoma/imunologia , Microambiente Tumoral/imunologia , Imunidade Adaptativa , Células Dendríticas/imunologia , Humanos , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Células Supressoras Mieloides/imunologia , Neutrófilos/imunologia , Prognóstico , Evasão Tumoral/imunologiaRESUMO
The molecular complexity of human breast cancer (BC) renders the clinical management of the disease challenging. Long non-coding RNAs (lncRNAs) are promising biomarkers for BC patient stratification, early detection, and disease monitoring. Here, we identified the involvement of the long intergenic non-coding RNA 01087 (LINC01087) in breast oncogenesis. LINC01087 appeared significantly downregulated in triple-negative BCs (TNBCs) and upregulated in the luminal BC subtypes in comparison to mammary samples from cancer-free women and matched normal cancer pairs. Interestingly, deregulation of LINC01087 allowed to accurately distinguish between luminal and TNBC specimens, independently of the clinicopathological parameters, and of the histological and TP53 or BRCA1/2 mutational status. Moreover, increased expression of LINC01087 predicted a better prognosis in luminal BCs, while TNBC tumors that harbored lower levels of LINC01087 were associated with reduced relapse-free survival. Furthermore, bioinformatics analyses were performed on TNBC and luminal BC samples and suggested that the putative tumor suppressor activity of LINC01087 may rely on interferences with pathways involved in cell survival, proliferation, adhesion, invasion, inflammation and drug sensitivity. Altogether, these data suggest that the assessment of LINC01087 deregulation could represent a novel, specific and promising biomarker not only for the diagnosis and prognosis of luminal BC subtypes and TNBCs, but also as a predictive biomarker of pharmacological interventions.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células MCF-7 , Metástase Neoplásica , Recidiva Local de Neoplasia , Intervalo Livre de Progressão , Mapas de Interação de Proteínas , RNA Longo não Codificante/genética , Transdução de Sinais , Fatores de Tempo , Transcriptoma , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Effector T cells (TEFF) are a barrier to booster vaccination because they can rapidly kill Ag-bearing APCs before memory T cells are engaged. We report in this study that i.v. delivery of rhabdoviral vectors leads to direct infection of follicular B cells in the spleen, where the earliest evidence of secondary T cell responses was observed. This allows booster immunizations to rapidly expand CD8(+) central memory T cells (TCM) during the acute phase of the primary response that is dominated by TEFF Interestingly, although the ablation of B cells before boosting with rhabdoviral vectors diminishes the expansion of memory T cells, B cells do not present Ags directly. Instead, depletion of CD11c(+) dendritic cells abrogates secondary T cell expansion, suggesting that virus-infected follicular B cells may function as an Ag source for local DCs to subsequently capture and present the Ag. Because TCM are located within B cell follicles in the spleen whereas TEFF cannot traffic through follicular regions, Ag production and presentation by follicular APCs represent a unique mechanism to secure engagement of TCM during an ongoing effector response. Our data offer insights into novel strategies for rapid expansion of CD8(+) T cells using prime-boost vaccines by targeting privileged sites for Ag presentation.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas Foliculares/imunologia , Baço/citologia , Baço/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vacinas Virais/imunologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Hepatitis B splicing-regulated protein (HBSP) of the hepatitis B virus (HBV) was uncovered a few years ago but its function remains unknown. HBSP expression occurs from a spliced viral transcript that increases during the course of liver disease. This study aimed at characterizing the impact of HBSP on cellular signaling pathways in vitro and on liver pathogenesis in transgenic (Tg) mice. By RT-qPCR array, NF-κB-inducible genes appeared modulated in HepG2 cells transduced with a HBSP-encoding lentivirus. Using luciferase and Western blot assays, we observed a decreased activation of the NF-κB pathway in HBSP-expressing cells following TNF-α treatment, as illustrated by lower levels of phosphorylated IκB-α. Meanwhile, the level of phosphorylated JNK increased together with the sensitivity to apoptosis. The contrasting effects on JNK and IκB-α activation upon TNF-α stimulation matched with a modulated maturation of TGF-ß-activated kinase 1 (TAK1) kinase, assessed by 2-dimensional SDS-PAGE. Inhibition of the NF-κB pathway by HBSP was confirmed in the liver of HBSP Tg mice and associated with a significant decrease of chemically induced chronic liver inflammation, as assessed by immunohistochemistry. In conclusion, HBSP contributes to limit hepatic inflammation during chronic liver disease and may favor HBV persistence by evading immune response.
Assuntos
Processamento Alternativo , Carcinoma Hepatocelular/metabolismo , Hepatite B/metabolismo , Inflamação/prevenção & controle , Neoplasias Hepáticas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Virais/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Proliferação de Células , Perfilação da Expressão Gênica , Hepatite B/imunologia , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/metabolismo , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas Virais/metabolismoRESUMO
Rapid boosting of memory CD8(+) T cells (TM) is essential in cancer immunotherapy and the control of certain infectious diseases. However, effector T cells (TE) are a barrier to booster vaccination because they can rapidly kill antigen-bearing antigen-presenting cells (APCs) before TM are engaged. We demonstrate that viral-vectored vaccines delivered by B cells elicit robust TM expansion in the presence of TE, enabling booster immunizations to bypass TE-mediated negative feedback regulation. Our data indicate that viral vector-loaded B cells home to the follicular regions in secondary lymphoid organs, which are anatomically separated from TE and in close proximity to TM. The B cells, however, do not serve as APCs in this area. Rather, classic CD11c(+) dendritic cells serve to stimulate the secondary CD8(+) T-cell response. Our data reveal that B cells represent a novel and readily accessible delivery system that can effectively engage secondary CD8(+) T-cell activation for prime-boost strategies.
Assuntos
Adenoviridae , Linfócitos B/transplante , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/administração & dosagem , Memória Imunológica , Imunoterapia Adotiva/métodos , Aceleração , Adenoviridae/genética , Animais , Linfócitos B/metabolismo , Células Cultivadas , Técnicas de Transferência de Genes , Imunização Secundária/métodos , Memória Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vacinação/métodosRESUMO
Within the oncolytic virus field, the extent of virus replication that is essential for immune stimulation to control tumor growth remains unresolved. Using infected cell protein 0 (ICP0)-defective oncolytic Herpes simplex virus type 1 (HSV-1) and HSV-2 viruses (dICP0 and dNLS) that show differences in their in vitro replication and cytotoxicity, we investigated the inherent features of oncolytic HSV viruses that are required for potent antitumor activity. In vitro, the HSV-2 vectors showed rapid cytotoxicity despite lower viral burst sizes compared to HSV-1 vectors. In vivo, although both of the dICP0 vectors initially replicated to a similar level, HSV-1 dICP0 was rapidly cleared from the tumors. In spite of this rapid clearance, HSV-1 dICP0 treatment conferred significant survival benefit. HSV-1 dICP0-treated tumors showed significantly higher levels of danger-associated molecular patterns that correlated with higher numbers of antigen-presenting cells within the tumor and increased antigen-specific CD8+ T-cell levels in the peripheral blood. This study suggests that, at least in the context of oncolytic HSV, the initial stages of immunogenic virus replication leading to activation of antitumor immunity are more important than persistence of a replicating virus within the tumor. This knowledge provides important insight for the design of therapeutically successful oncolytic viruses.
Assuntos
Vetores Genéticos/genética , Neoplasias/genética , Neoplasias/imunologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Simplexvirus/genética , Simplexvirus/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Apoptose/genética , Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Proteína HMGB1/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/imunologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Mutação , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica , Receptor ErbB-2/imunologia , Carga Tumoral/genética , Carga Tumoral/imunologia , Replicação ViralRESUMO
The rhabdovirus Maraba has recently been characterized as a potent oncolytic virus. In the present study, we engineered an attenuated Maraba strain, defined as MG1, to express a melanoma-associated tumor antigen. Its ability to mount an antitumor immunity was evaluated in tumor-free and melanoma tumor-bearing mice. Alone, the MG1 vaccine appeared insufficient to prime detectable adaptive immunity against the tumor antigen. However, when used as a boosting vector in a heterologous prime-boost regimen, MG1 vaccine rapidly generated strong antigen-specific T-cell immune responses. Once applied for treating syngeneic murine melanoma tumors, our oncolytic prime-boost vaccination protocol involving Maraba MG1 dramatically extended median survival and allowed complete remission in more than 20% of the animals treated. This work describes Maraba virus MG1 as a potent vaccine vector for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive antitumor immunity.
Assuntos
Vetores Genéticos/genética , Vírus Oncolíticos/genética , Rhabdoviridae/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Efeito Citopatogênico Viral , Feminino , Expressão Gênica , Vetores Genéticos/imunologia , Imunização Secundária/métodos , Oxirredutases Intramoleculares/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Melanoma Experimental , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Vírus Oncolíticos/imunologia , Rhabdoviridae/imunologia , Resultado do Tratamento , Vesiculovirus/genética , Vesiculovirus/imunologia , Tropismo ViralRESUMO
Histone deacetylase inhibitors (HDACi) can modulate innate antiviral responses and render tumors more susceptible to oncolytic viruses (OVs); however, their effects on adaptive immunity in this context are largely unknown. Our present study reveals an unexpected property of the HDACi MS-275 that enhances viral vector-induced lymphopenia leading to selective depletion of bystander lymphocytes and regulatory T cells while allowing expansion of antigen-specific secondary responses. Coadministration of vaccine plus drug during the boosting phase focuses the immune response on the tumor by suppressing the primary immune response against the vaccine vector and enhancing the secondary response against the tumor antigen. Furthermore, improvement of T cell functionality was evident suggesting that MS-275 can orchestrate a complex array of effects that synergize immunotherapy and viral oncolysis. Surprisingly, while MS-275 dramatically enhanced efficacy, it suppressed autoimmune pathology, profoundly improving the therapeutic index.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Imunoterapia/métodos , Neoplasias/terapia , Animais , Autoimunidade/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Melanoma/tratamento farmacológico , Melanoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológicoRESUMO
Tigilanol tiglate is an oncolytic small molecule that is undergoing clinical trials. A recent study revealed the capacity of this pyroptosis inducer to elicit hallmarks of immunogenic cell death. In addition, intratumoral injection of tigilanol tiglate can sensitize subcutaneous cancers to subsequent immune checkpoint inhibitors targeting CTLA-4 alone or in combination with PD-1.
Assuntos
Neoplasias , Humanos , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Animais , Inibidores de Checkpoint Imunológico/administração & dosagem , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Morte Celular Imunogênica/efeitos dos fármacos , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Piroptose/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologiaRESUMO
Doxorubicin is a prototypical inducer of immunogenic cell death (ICD) that sensitizes to subsequent immunotherapy by PD-1 blockade. However, this systemic drug combination fails against glioblastoma, hidden behind the blood-brain barrier (BBB). A recent work delineates a biophysical method for BBB permeabilization that yields effective preclinical effects of chemoimmunotherapy.
Assuntos
Barreira Hematoencefálica , Neoplasias Encefálicas , Doxorrubicina , Glioblastoma , Imunoterapia , Receptor de Morte Celular Programada 1 , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Glioblastoma/imunologia , Humanos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Doxorrubicina/administração & dosagem , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Imunoterapia/métodos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologiaRESUMO
Advances in single-cell RNA and T cell receptor (TCR) sequencing allow to study the specificity and functionality of tumor-infiltrating T lymphocytes. A recent study unravels fundamental differences between microsatellite-instable (MSI) colorectal cancers, in which T cells tend to be tumor-specific, and microsatellite-stable (MSS) cancers, in which T cells exhibit bystander features.
Assuntos
Neoplasias Colorretais , Linfócitos T , Humanos , Análise de Célula Única , Linfócitos do Interstício Tumoral , Instabilidade de Microssatélites , Neoplasias Colorretais/genéticaRESUMO
Oncolytic viruses (OVs) represent a novel class of cancer immunotherapy agents that preferentially infect and kill cancer cells and promote protective antitumor immunity. Furthermore, OVs can be used in combination with established or upcoming immunotherapeutic agents, especially immune checkpoint inhibitors, to efficiently target a wide range of malignancies. The development of OV-based therapy involves three major steps before clinical evaluation: design, production and preclinical testing. OVs can be designed as natural or engineered strains and subsequently selected for their ability to kill a broad spectrum of cancer cells rather than normal, healthy cells. OV selection is further influenced by multiple factors, such as the availability of a specific viral platform, cancer cell permissivity, the need for genetic engineering to render the virus non-pathogenic and/or more effective and logistical considerations around the use of OVs within the laboratory or clinical setting. Selected OVs are then produced and tested for their anticancer potential by using syngeneic, xenograft or humanized preclinical models wherein immunocompromised and immunocompetent setups are used to elucidate their direct oncolytic ability as well as indirect immunotherapeutic potential in vivo. Finally, OVs demonstrating the desired anticancer potential progress toward translation in patients with cancer. This tutorial provides guidelines for the design, production and preclinical testing of OVs, emphasizing considerations specific to OV technology that determine their clinical utility as cancer immunotherapy agents.
RESUMO
Cholangiocarcinoma (CCA) is a malignancy affecting the epithelial cells that line the bile ducts. This cancer shows a poor prognosis and current treatments remain inefficient. Orthotopic CCA mouse models are useful for the development of innovative therapeutic strategies. Here, we describe an orthotopic model of intrahepatic CCA that can be easily induced in mice within 5 weeks at a high incidence. It is achieved by expressing two oncogenes, namely, (i) the intracellular domain of the Notch1 receptor (NICD) and (ii) AKT, in hepatocytes by means of the sleeping beauty transposon system. These plasmid vectors are delivered by hydrodynamic injection into the tail vein. The present chapter also describes how to perform magnetic resonance imaging (MRI) of the livers to visualize intrahepatic CCA nodules.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Camundongos , Animais , Ductos Biliares Intra-Hepáticos , Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/genética , Oncogenes/genética , Fígado/patologiaRESUMO
Tissue-resident and recruited immune cells are essential mediators of natural and therapy-induced immunosurveillance of liver neoplasia. This idea has been recently reinforced by the clinical approval of immune checkpoint inhibitors for the immunotherapy of hepatocellular carcinoma and cholangiocarcinoma. Such research progress relies on the in-depth characterization of the immune populations that are present in pre-neoplastic and neoplastic hepatic lesions. A convenient technology for advancing along this path is high-dimensional cytometry.In this chapter, we present a protocol to assess the subtype and differentiation state of hepatic lymphocyte populations by multicolor immunofluorescence staining and flow cytometry. We detail the steps required for viability assessment and immune cell phenotyping of single-cell suspensions of liver cells by means of surface and intracellular staining of more than a dozen markers of interest. This protocol does not require prior removal of debris and dead cells and allows to process multiple samples in parallel. The procedure includes the use of a fixative-resistant viability dye that allows cell fixation and permeabilization after cell surface staining and before intracellular staining and data acquisition on a flow cytometer. Moreover, we provide a panel of fluorochrome-labeled antibodies designed for the characterization of lymphocytic subsets that can be adapted to distinct experimental settings. Finally, we present an overview of the post-staining pipeline, including data acquisition on a flow cytometer and tools for post-acquisition analyses.