RESUMEN
Melanoma has been a prototype for cancer immunology research, and the mechanisms of anti-tumor T-cell responses have been extensively investigated in patients treated with various immunotherapies. Individual differences in cancer-immune status are defined mainly by cancer cell characteristics such as DNA mutations generating immunogenic neo-antigens, and oncogene activation causing immunosuppression, but also by patients' genetic backgrounds such as HLA types and genetic polymorphisms of immune related molecules, and environmental and lifestyle factors such as UV rays, smoking, gut microbiota and concomitant medications; these factors have an influence on the efficacy of immunotherapy. Recent comparative studies on responders and non-responders in immune-checkpoint inhibitor therapy using various new technologies including multi-omics analyses on genomic DNA, mRNA, metabolites and microbiota and single cell analyses of various immune cells have led to the advance of human tumor immunology and the development of new immunotherapy. Based on the new findings from these investigations, personalized cancer immunotherapies along with appropriate biomarkers and therapeutic targets are being developed for patients with melanoma. Here, we will discuss one of the essential subjects in tumor immunology: identification of immunogenic tumor antigens and their effective use in various immunotherapies including cancer vaccines and adoptive T-cell therapy.
Asunto(s)
Vacunas contra el Cáncer , Melanoma , Humanos , Linfocitos T , Antígenos Específicos del Melanoma , Melanoma/tratamiento farmacológico , Inmunoterapia , Antígenos de Neoplasias , Vacunas contra el Cáncer/uso terapéutico , Inmunoterapia AdoptivaRESUMEN
One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and understanding their molecular mechanisms for future use in clinical settings. We previously identified Macrophage Migration Inhibitory Factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs based on in vitro functional cloning strategy and revealed that MIF can support the proliferation of human brain tumor-initiating cells (BTICs). However, the detailed downstream signaling for the functions has largely remained unknown. Thus, in the present study, we newly identified translationally-controlled tumor protein-1 (TPT1), which is expressed in the ventricular zone of mouse embryonic brain, as a downstream target of MIF signaling in mouse and human NSPCs and human BTICs. Using gene manipulation (over or downregulation of TPT1) techniques including CRISPR/Cas9-mediated heterozygous gene disruption showed that TPT1 contributed to the regulation of cell proliferation/survival in mouse NSPCs, human embryonic stem cell (hESC) derived-NSPCs, human-induced pluripotent stem cells (hiPSCs) derived-NSPCs and BTICs. Furthermore, gene silencing of TPT1 caused defects in neuronal differentiation in the NSPCs in vitro. We also identified the MIF-CHD7-TPT1-SMO signaling axis in regulating hESC-NSPCs and BTICs proliferation. Intriguingly, TPT1suppressed the miR-338 gene, which targets SMO in hESC-NSPCs and BTICs. Finally, mice with implanted BTICs infected with lentivirus-TPT1 shRNA showed a longer overall survival than control. These results also open up new avenues for the development of glioma therapies based on the TPT1 signaling pathway.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos , Células Madre Neoplásicas , Células-Madre Neurales , Proteína Tumoral Controlada Traslacionalmente 1 , Animales , Encéfalo/metabolismo , Proliferación Celular/fisiología , Humanos , Oxidorreductasas Intramoleculares , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Ratones , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Células-Madre Neurales/metabolismo , Proteína Tumoral Controlada Traslacionalmente 1/genéticaRESUMEN
Modulation of the immunosuppressive tumor microenvironment (TME) is essential for enhancing the anti-tumor effects of immune checkpoint inhibitors (ICIs). Adhesion molecules and enzymes such as vascular adhesion protein-1 (VAP-1), which are expressed in some cancers and tumor vascular endothelial cells, may be involved in the generation of an immunosuppressive TME. In this study, the role of VAP-1 in TME was investigated in 2 murine colon cancer models and human cancer cells. Intraperitoneal administration of the VAP-1-specific inhibitor U-V296 inhibited murine tumor growth by enhancing IFN-γ-producing tumor antigen-specific CD8+ T cells. U-V296 exhibited significant synergistic anti-tumor effects with ICIs. In the TME of mice treated with U-V296, the expression of genes associated with M2-like macrophages, Th2 cells (Il4, Retnla, and Irf4), angiogenesis (Pecam1), and fibrosis (Acta2, Loxl2) were significantly decreased, and the Th1/Th2 balance was increased. H2 O2 , an enzymatic product of VAP-1, which promoted the production of IL-4 by mouse Th2 and inhibited IFN-γ by mouse Th1 and human tumor-infiltrating lymphocytes, was decreased in tumors and CD31+ tumor vascular endothelial cells in the TMEs of mice treated with VAP-1 inhibitor. TCGA database analysis showed that VAP-1 expression was a negative prognostic factor in human cancers, exhibiting a significant positive correlation with IL-4, IL4R, and IL-13 expression and a negative correlation with IFN-γ expression. These results indicated that VAP-1 is involved in the immunosuppressive TMEs through H2 O2 -associated Th2/M2 conditions and may be an attractive target for the development of combination cancer immunotherapy with ICIs.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Moléculas de Adhesión Celular/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias/inmunología , Neoplasias/terapia , Amina Oxidasa (conteniendo Cobre)/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Moléculas de Adhesión Celular/inmunología , Línea Celular Tumoral , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Femenino , Inmunoterapia/mortalidad , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
To evaluate the feasibility of adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) in Japanese patients with melanoma who failed immune-checkpoint inhibitor therapy, an open-label, single-arm, pilot study was conducted. We investigated the immunological and genetic factors of the pretreatment tumor and expanded TILs that may be associated with the clinical response. The treatment protocol comprised preparation of TIL culture, lympho-depleting non-myeloablative preconditioning with cyclophosphamide and fludarabine, TIL infusion, and intravenous administration of low-dose IL-2. Three patients of clinical subtypes mucosal, superficial spreading, and acral melanoma underwent TIL-ACT. Most severe adverse events, including fever and leukopenia, were manageable with the supportive regimen specified in the protocol, suggesting that the TIL-ACT regimen is suitable for Japanese patients with melanoma. One patient showed a short-term partial response, one relatively long-stable disease, and one experienced disease progression. Whole-exome and transcriptional sequencing of isolated tumor cells and immunohistochemical analyses before TIL-ACT revealed various immunostimulatory factors, including a high tumor mutation burden and immune cell-recruiting chemokines, as well as various immunosuppressive factors including TGF-ß, VEGF, Wnt/ß-catenin, and MAPK signaling and epithelial-to-mesenchymal transition, which might influence the efficacy of TIL-ACT. Our results imply mechanisms for the antitumor effect of and resistance to TIL-ACT. Further studies of immune-resistant mechanisms of TIL-ACT are warranted. This study is registered with the UMIN Clinical Trial Registry (UMIN 000011431).
Asunto(s)
Ciclofosfamida/administración & dosificación , Interleucina-2/administración & dosificación , Linfocitos Infiltrantes de Tumor/trasplante , Melanoma/terapia , Vidarabina/análogos & derivados , Administración Intravenosa , Técnicas de Cultivo de Célula , Ciclofosfamida/uso terapéutico , Estudios de Factibilidad , Redes Reguladoras de Genes , Humanos , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia Adoptiva , Linfocitos Infiltrantes de Tumor/citología , Masculino , Melanoma/genética , Melanoma/inmunología , Persona de Mediana Edad , Proyectos Piloto , Acondicionamiento Pretrasplante , Resultado del Tratamiento , Vidarabina/administración & dosificación , Vidarabina/uso terapéuticoRESUMEN
Immune checkpoint inhibitors (ICI) such as PD-1/PD-L1 antibodies (Abs) and CTLA4 Abs and T cell-based adoptive cell therapies are effective for patients with various cancers. However, response rates of ICI monotherapies are still limited due to lack of immunogenic antigens and various immune-resistant mechanisms. The latter includes adaptive immune resistance that is caused by anti-tumor T cells (e.g. PD-L1 induced by IFN-γ from T cells) and primary immune resistance that is caused by cancer cells (e.g. immunosuppressive cytokines produced by cancer cells). Further understanding of the immune-resistant mechanisms, which may be possible through comparative analyses of responders and non-responders to the immunotherapies, will lead to the identification of new diagnostic biomarkers and therapeutic targets for development of effective cancer immuno therapies.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Resistencia a Antineoplásicos/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Inmunidad Adaptativa , Antígeno B7-H1/inmunología , Antígeno CTLA-4/inmunología , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Inmunoterapia , Neoplasias/patología , Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: To determine the involvement of homeobox D9 (HOXD9) in the survival, proliferation, and metastasis of cervical cancer cells through regulating the expression of human papillomavirus (HPV) 16 E6/E7 genes using the P97 promoter. METHODS: One hundred cases of cervical cancer (CC), CC cell lines SKG-I, SKG-II, SKG-IIIa, SKG-IIIb, HeLa, and SiHa, and a human tumor xenograft mouse model were used to examine the roles of HOXD9 in CC. Knockdown experiments employed RNA interference of HOXD9. qPCR, functional assays, western blotting, DNA microarray, and luciferase and ChIP assays were applied for assessments. RESULTS: All CC cell lines expressed HOXD9 mRNA and protein. In uterine CC, HOXD9 gene expression was significantly higher than in normal cervical tissues. A positive correlation of lymphovascular space invasion and lymph node metastasis with high levels of HOXD9 expression was found in patient samples. HOXD9-knockdown cells in the mouse xenograft model only formed small or no tumors. Knockdown of HOXD9 markedly reduced CC cell proliferation, migration and invasion, induced apoptosis, increased P53 protein expression, and suppressed HPV E6/E7 expression by directly binding to the P97 promoter of HPV16 E6/E7 genes. A positive correlation between HOXD9 and HPV16 E6 expression was found in CC patients. CONCLUSIONS: HOXD9 promotes HPV16 E6 and E7 expression by direct binding to the P97 promoter, which enhances proliferation, migration, and metastasis of CCr cells. Our results suggest that HOXD9 could be a prognostic biomarker and potential therapeutic target in CC.
Asunto(s)
Proteínas de Homeodominio/fisiología , Proteínas de Neoplasias/fisiología , Infecciones por Papillomavirus/genética , Regiones Promotoras Genéticas/genética , Neoplasias del Cuello Uterino/virología , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Papillomavirus Humano 16/genética , Humanos , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Proteínas Oncogénicas Virales/metabolismo , Oncogenes , Proteínas E7 de Papillomavirus/metabolismo , Fenotipo , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/genéticaRESUMEN
Kinesin family member 20A (KIF20A), an ideal cancer-testis antigen, was reported to be a promising immunotherapeutic target for pancreatic cancers. Clinical trials of KIF20A peptide vaccine immunotherapy have been conducted against pancreatic cancers. To demonstrate the efficacy of KIF20A as a candidate molecular target for gliomas, we analyzed the expression and function of KIF20A in gliomas. Western blot and quantitative PCR analyses showed that KIF20A expression in glioma cell lines and glioma tissues was high compared with that found in a normal brain. KIF20A immunostaining of glioma cells and glioma tissues demonstrated that KIF20A was involved in spindle formation and cytokinesis, and that KIF20A was highly expressed, especially in glioma cells undergoing mitosis. In silico analysis of a cancer microarray database revealed that KIF20A was highly expressed in gliomas depending on the pathological grade, and glioma patients with higher expression of KIF20A showed poorer prognosis. Down-regulating KIF20A reduced cell proliferation in glioma cells due to the failure of cytokinesis and generation of binucleated cells. Additionally, KIF20A inhibition induced significant apoptosis in SF126 glioma cells. Taken together, KIF20A is a tumor-associated antigen involved in the glioma cell growth and cell survival, suggesting that KIF20A is an oncoantigen of gliomas. Thus, KIF20A is a candidate novel immunotherapeutic target for gliomas.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Glioma/inmunología , Glioma/metabolismo , Cinesinas/metabolismo , Apoptosis , Encéfalo/metabolismo , Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular , Humanos , InmunoterapiaRESUMEN
In a previous study, we showed that murine dendritic cells (DCs) can increase the number of neural stem/progenitor cells (NSPCs) in vitro and in vivo. In the present study, we identified macrophage migration inhibitory factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs in vitro. MIF is secreted by DCs and NSPCs, and its function in the normal brain remains largely unknown. It was previously shown that in macrophages, MIF binds to a CD74-CD44 complex. In the present study, we observed the expression of MIF receptors in mouse ganglionic-eminence-derived neurospheres using flow cytometry in vitro. We also found CD74 expression in the ganglionic eminence of E14 mouse brains, suggesting that MIF plays a physiological role in vivo. MIF increased the number of primary and secondary neurospheres. By contrast, retrovirally expressed MIF shRNA and MIF inhibitor (ISO-1) suppressed primary and secondary neurosphere formation, as well as cell proliferation. In the neurospheres, MIF knockdown by shRNA increased caspase 3/7 activity, and MIF increased the phosphorylation of Akt, Erk, AMPK and Stat3 (Ser727), as well as expression of Hes3 and Egfr, the products of which are known to support cell survival, proliferation and/or maintenance of NSPCs. MIF also acted as a chemoattractant for NSPCs. These results show that MIF can induce NSPC proliferation and maintenance by multiple signaling pathways acting synergistically, and it may be a potential therapeutic factor, capable of activating NSPC, for the treatment of degenerative brain disorders.
Asunto(s)
Proliferación Celular , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Células-Madre Neurales/citología , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Recuento de Células , Movimiento Celular , Supervivencia Celular , Células Cultivadas , Clonación Molecular , Células Dendríticas/citología , Células Dendríticas/metabolismo , Activación Enzimática , Femenino , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunohistoquímica , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/farmacología , Lentivirus/genética , Lentivirus/metabolismo , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/farmacología , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Fosforilación , Embarazo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de SeñalRESUMEN
Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4(+) HNSCC cells, but not CCR4(-) cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206(+) M2-like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206(high) M2-like macrophages increased the cell motility of CCR4(+) HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4(+) cancer cells, but not CCR4(-) cells, metastasized to lymph nodes which contained CCL22 producing M2-like macrophages. These results demonstrate that lymph node metastasis of CCR4(+) HNSCC is promoted by CCL22 in an autocrine or M2-like macrophage-dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Comunicación Celular , Quimiocina CCL22/metabolismo , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores CCR4/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Comunicación Autocrina , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Neoplasias de Cabeza y Cuello/patología , Humanos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Ratones , Persona de Mediana Edad , Comunicación Paracrina , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
To identify therapeutic molecular targets for glioma, we performed modified serological identification of antigens by recombinant complementary DNA (cDNA) expression cloning using sera from a mouse glioma model. Two clones, kinesin family member 23 (Kif23) and structural maintenance of chromosomes 4 (Smc4), were identified as antigens through immunological reaction with sera from mice harboring synergic GL261 mouse glioma and intratumoral inoculation with a mutant herpes simplex virus. The human Kif23 homolog KIF23 is a nuclear protein that localizes to the interzone of mitotic spindles, acting as a plus-end-directed motor enzyme that moves antiparallel microtubules in vitro. Expression analysis revealed a higher level of KIF23 expression in glioma tissues than in normal brain tissue. The introduction of small interfering RNA (siRNA) targeting KIF23 into two different glioma cell lines, U87MG and SF126, downregulated KIF23 expression, which significantly suppressed glioma cell proliferation in vitro. KIF23 siRNA-treated glioma cells exhibited larger cell bodies with two or more nuclei compared with control cells. In vivo analysis using mouse xenograft showed that KIF23 siRNA/DNA chimera-treated tumors were significantly smaller than tumors treated with control siRNA/DNA chimera. Taken together, our results indicate that downregulation of KIF23 decreases proliferation of glioma cells and that KIF23 may be a novel therapeutic target in malignant glioma.
Asunto(s)
Neoplasias Encefálicas/patología , Proliferación Celular , Regulación hacia Abajo/fisiología , Glioma/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Antígeno Ki-67/metabolismo , Ratones , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/genética , Mutación/genética , Invasividad Neoplásica , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Simplexvirus/inmunología , Factores de Tiempo , Transfección , Trasplante Heterólogo/métodosRESUMEN
Cancer stem cells are relatively resistant to chemotherapy, and cause relapse of cancer. Thus, various strategies to eliminate cancer stem cells have recently been exploited. One of them is immunotherapy. To develop the immunotherapy targeting cancer stem cells, tumor antigens expressed in cancer stem cells have been identified, and their use in the immunotherapy is expected. However, cancer stem cells may have an immunosuppressive ability. Therefore, blockade of the immunosuppressive mechanisms of cancer stem cells may also be required for development of effective immunotherapies against cancer stem cells.
Asunto(s)
Inmunoterapia/métodos , Neoplasias/terapia , Células Madre Neoplásicas/inmunología , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Humanos , Terapia de Inmunosupresión/efectos adversos , Neoplasias/inmunología , Células Madre Neoplásicas/efectos de los fármacosRESUMEN
BACKGROUND: Understanding the mechanisms of non-T cell inflamed tumor microenvironment (TME) and their modulation are important to improve cancer immunotherapies such as immune checkpoint inhibitors. The involvement of various immunometabolisms has recently been indicated in the formation of immunosuppressive TME. In this study, we investigated the immunological roles of stearoyl-CoA desaturase 1 (SCD1), which is essential for fatty acid metabolism, in the cancer immune response. METHODS: We investigated the roles of SCD1 by inhibition with the chemical inhibitor or genetic manipulation in antitumor T cell responses and the therapeutic effect of anti-programmed cell death protein 1 (anti-PD-1) antibody using various mouse tumor models, and their cellular and molecular mechanisms. The roles of SCD1 in human cancers were also investigated by gene expression analyses of colon cancer tissues and by evaluating the related free fatty acids in sera obtained from patients with non-small cell lung cancer who were treated with anti-PD-1 antibody. RESULTS: Systemic administration of a SCD1 inhibitor in mouse tumor models enhanced production of CCL4 by cancer cells through reduction of Wnt/ß-catenin signaling and by CD8+ effector T cells through reduction of endoplasmic reticulum stress. It in turn promoted recruitment of dendritic cells (DCs) into the tumors and enhanced the subsequent induction and tumor accumulation of antitumor CD8+ T cells. SCD1 inhibitor was also found to directly stimulate DCs and CD8+ T cells. Administration of SCD1 inhibitor or SCD1 knockout in mice synergized with an anti-PD-1 antibody for its antitumor effects in mouse tumor models. High SCD1 expression was observed in one of the non-T cell-inflamed subtypes in human colon cancer, and serum SCD1 related fatty acids were correlated with response rates and prognosis of patients with non-small lung cancer following anti-PD-1 antibody treatment. CONCLUSIONS: SCD1 expressed in cancer cells and immune cells causes immunoresistant conditions, and its inhibition augments antitumor T cells and therapeutic effects of anti-PD-1 antibody. Therefore, SCD1 is an attractive target for the development of new diagnostic and therapeutic strategies to improve current cancer immunotherapies including immune checkpoint inhibitors.
Asunto(s)
Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias del Colon , Neoplasias Pulmonares , Estearoil-CoA Desaturasa , Animales , Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Ratones , Ratones Noqueados , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Estearoil-CoA Desaturasa/inmunología , Microambiente Tumoral , Vía de Señalización Wnt/inmunología , beta Catenina/inmunologíaRESUMEN
BACKGROUND: HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies. RESULTS: In this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs). CONCLUSIONS: Our results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.
Asunto(s)
Neoplasias Encefálicas/fisiopatología , Regulación Neoplásica de la Expresión Génica , Glioma/fisiopatología , Proteínas de Homeodominio/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/patología , Apoptosis/genética , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Proliferación Celular , Análisis por Conglomerados , Perfilación de la Expresión Génica , Silenciador del Gen , Glioma/genética , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Proteínas de Neoplasias/genéticaRESUMEN
Use of adequate adjuvant is necessary for induction of effective antitumor immune responses. To develop an effective adjuvant for cancer immunotherapy, we selected formalin-inactivated (f)-HSV as an adjuvant component, and analyzed the mechanisms underlying its adjuvant effects. First, we found that f-HSV can induce the tumor antigen-specific CTLs by enhancing antigen cross-presentation by dendritic cells (DCs), mainly through TLR2, but not TLR9. Next, f-HSV was also found to prevent the accumulation of myeloid-derived suppressor cells (MDSCs). We demonstrated that the expansion of MDSCs in the blood and spleen during tumor progression required B cells producing the inflammatory angiogenesis factors, vascular endothelial growth factor (VEGF)-A and neuropilin-1 (NRP-1), a co-receptor for VEGF receptor-2 (VEGFR-2). Interestingly, the transmembrane-type NRP-1 on B cells changed to soluble-type NRP-1 (sNRP-1) by f-HSV treatment. We further showed that the sNRP-1 and VEGF-A secreted from B cells by f-HSV treatment could abrogate the immunosuppressive ability of MDSCs. These results suggest that f-HSV can enhance antitumor immune responses as an adjuvant, not only through activation of DCs, but also inactivation of MDSCs via B cells.
Asunto(s)
Células Dendríticas/inmunología , Inmunoterapia/métodos , Células Mieloides/inmunología , Neoplasias Experimentales/terapia , Simplexvirus/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Tumoral , Chlorocebus aethiops , Reactividad Cruzada/inmunología , Femenino , Formaldehído , Inmunización/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Linfocitos T Citotóxicos/inmunología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Células VeroRESUMEN
To identify molecular therapeutic targets for glioma, we performed gene expression profiling by using a complementary DNA (cDNA) microarray method and identified the urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180) as a gene expressed highly in glioma tissue compared with the normal brain tissue. The uPARAP is an endocytic receptor for collagen. In certain cell types, uPARAP occurs in a complex with the urokinase plasminogen activator receptor (uPAR) where it fulfills other functions in addition to collagenolysis. Quantitative PCR analysis using a cDNA panel revealed higher expression levels of uPARAP in glioma tissue compared with normal brain tissue. Western blot analysis revealed that the uPARAP protein was expressed in glioma samples and two glioma cell lines, KNS42 and KNS81, but not expressed in control tissue from the normal brain. Introduction of small interfering RNA-targeted uPARAP into the two different glioma cell lines, KNS42 and KNS81, resulted in downregulation of uPARAP expression, and it significantly suppressed glioma cell migration and invasion in vitro. Control glioma cells showed small cell bodies, whereas uPARAP siRNA-treated glioma cells exhibited large and flat morphology. Most of the polymeric actin in the control glioma cells was concentrated in the lamellipodia that are observed in mobile cells. In contrast, in the uPARAP siRNA-treated glioma cells, polymeric actin became organized in stress fibers and the lamellipodia disappeared, characteristic of immobile cells. Our present study suggests that uPARAP may be involved in glioma cell invasiveness through actin cytoskeletal rearrangement. downregulation of uPARAP may be a novel anti-invasion therapeutic strategy for malignant gliomas.
Asunto(s)
Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Glioma/metabolismo , Lectinas de Unión a Manosa/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Acetilcisteína , Western Blotting , Línea Celular Tumoral , Citoesqueleto/patología , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Perfilación de la Expresión Génica , Glioma/genética , Glioma/patología , Humanos , Lectinas de Unión a Manosa/genética , Glicoproteínas de Membrana/genética , Invasividad Neoplásica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/genéticaRESUMEN
Objective: Brain tumor-initiating cells are characterized by their features of self-renewal, multi-lineage differentiation, and tumorigenicity. We analyzed the gene expression of brain tumor-initiating cells to identify their novel cellular markers. Methods: We performed cDNA microarray, in silico expressed sequence tags (ESTs), RT-PCR, and q-PCR analyses. Results: We identified 10 genes that were more highly expressed in brain tumor-initiating cells than in neural stem cells. In addition, we identified 10 other genes that were more highly expressed in brain tumor-initiating cells than in glioma cell line cells from the cDNA microarray analysis. Using the EST database, we looked to see if the 20 genes were expressed more highly in gliomas, compared with normal adult brains. Among the 20 genes, five (KLRC2, HOXB2, KCNJ2, KLRC1, and COL20A1) were expressed more than twice in glioma samples, compared with normal adult brains, and, therefore, were referred for further evaluation. RT-PCR was conducted using cDNA samples obtained from neural stem cells, normal brain tissue, fetal brain tissue, glioma cell lines, and glioma tumor-initiating cell lines. KLRC2, a transmembrane activating receptor in natural killer cells, was expressed more highly in glioma-initiating cells than in neural stem cell lines or normal adult brain tissue. The q-PCR analysis revealed that expression of KLRC2 was significantly higher in brain tumor-initiating cells compared to normal brain controls. Conclusion: KLRC2 could be a novel cellular marker for brain tumor-initiating cells.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Subfamília C de Receptores Similares a Lectina de Células NK/genética , Células Madre Neoplásicas/citología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Glioma/patología , HumanosRESUMEN
Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c(+) cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.
Asunto(s)
Callithrix/inmunología , Células Dendríticas/inmunología , Animales , Células de la Médula Ósea/inmunología , Relación Dosis-Respuesta Inmunológica , Endocitosis/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Interferón gamma/biosíntesis , Interleucina-4/inmunología , Prueba de Cultivo Mixto de Linfocitos , Neurotrofina 3/biosíntesis , Proteínas Recombinantes/inmunología , Bazo/inmunología , Células Madre/inmunologíaRESUMEN
We have previously reported that the transplantation of dendritic cells (DCs) brings about functional recovery after spinal cord injury in mice through the activation of endogenous microglia/macrophages and neural stem/progenitor cells. In this study, the effect of interleukin-12 (IL-12), which is secreted from DCs, was evaluated for the treatment of spinal cord injury in mice. Administration of IL-12 into the injured site significantly increased the number of activated microglia/macrophages and DCs as well as the expression of brain-derived neurotrophic factor surrounding the lesion site. Immunohistochemical analyses showed that de novo neurogenesis and remyelination were induced by IL-12 treatment. Furthermore, an open field test using Basso-Beattie-Brenham scoring revealed a significant improvement of locomotor function in mice treated with IL-12. These results suggest that IL-12 administration into the injured spinal cord results in a functional recovery through the activation of microglia/macrophages and DCs.
Asunto(s)
Células Dendríticas/fisiología , Microglía/fisiología , Traumatismos de la Médula Espinal/rehabilitación , Animales , Bromodesoxiuridina , Femenino , Miembro Posterior , Inmunohistoquímica , Interleucina-12/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/fisiología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatologíaRESUMEN
To identify neuron-specific genes, we performed gene expression profiling, cDNA microarray and in silico ESTs (expressed sequence tags) analyses. We identified a human neuron-specific gene, KIAA1110 (homologue of rat synArfGEF (Po)), that is a member of the guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF). RT-PCR analysis showed that the KIAA1110 gene was expressed specifically in the brain among adult human tissues, whereas no apparent expression was observed in immature neural tissues/cells, such as fetal brain, glioma tissues/cells, and neural stem/precursor cells (NSPCs). The KIAA1110 protein was shown to be expressed in mature neurons but not in undifferentiated NSPCs. Immunohistochemical analysis also showed that KIAA1110 was expressed in neurons of the human adult cerebral cortex. Furthermore, the pull-down assay revealed that KIAA1110 has a GEF activity toward ARF1 that regulates transport along the secretion pathway. These results suggest that KIAA1110 is expressed specifically in mature neurons and may play an important role in the secretion pathway as a GEF for ARF1.