RESUMO
BACKGROUND: Approximately 20% of melanomas contain a mutation in NRAS. However no direct inhibitor of NRAS is available. One of the main signaling pathways downstream of NRAS is the MAPK pathway. In this study we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway. METHODS: Fourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated. RESULTS: The majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination. Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment. Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1. However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway. Resistant cell lines also showed higher levels of p-GSK3ß and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines. CONCLUSIONS: The combination of PRi + MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.
Assuntos
GTP Fosfo-Hidrolases/genética , Sistema de Sinalização das MAP Quinases/genética , Melanoma/genética , Proteínas de Membrana/genética , Mutação/genética , Quinases raf/genética , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D1/genética , Ciclina D3/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Transdução de Sinais/genética , Transcrição Gênica/genéticaRESUMO
BACKGROUND: In melanoma, dysregulation of the MAPK pathway, usually via BRAF(V600) or NRAS(Q61) somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially effective for BRAF-mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAF(V600), NRAS mutant, or wild-type melanoma. METHODS: The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined. RESULTS: Sensitivity fell into three groups: sensitive, 50% inhibitory concentration (IC50) < 1 µM; intermediately sensitive, IC50 1-2 µM; and resistant, >2 µM. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAF(V600) mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis. CONCLUSIONS: Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines and significantly delayed the onset of acquired resistance in long term in vitro assays. Therefore, SCH772984 may be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors.
Assuntos
GTP Fosfo-Hidrolases/antagonistas & inibidores , Indazóis/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Mieloma Múltiplo/patologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , GTP Fosfo-Hidrolases/genética , Humanos , Indóis/farmacologia , Concentração Inibidora 50 , Proteínas de Membrana/genética , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , VemurafenibRESUMO
Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to â¼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Citotoxinas/genética , Citotoxinas/uso terapêutico , Técnicas de Transferência de Genes , Terapia Genética , Glioma/tratamento farmacológico , Adenoviridae/genética , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Exotoxinas/genética , Exotoxinas/uso terapêutico , Vetores Genéticos/genética , Glioma/patologia , Humanos , Imunocompetência/imunologia , Interleucina-13/genética , Interleucina-13/uso terapêutico , Camundongos , Camundongos Nus , Mutação/genética , Neurotoxinas/toxicidade , Pseudomonas/metabolismo , Transgenes/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The temporospatial equilibrium of phosphate contributes to physiological bone development and fracture healing, yet optimal control of phosphate content has not been explored in skeletal regenerative materials. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) is a synthetic, tunable material that promotes in vivo skull regeneration. In this work, the effects of MC-GAG phosphate content on the surrounding microenvironment and osteoprogenitor differentiation are investigated. This study finds that MC-GAG exhibits a temporal relationship with soluble phosphate with elution early in culture shifting to absorption with or without differentiating primary bone marrow-derived human mesenchymal stem cells (hMSCs). The intrinsic phosphate content of MC-GAG is sufficient to stimulate osteogenic differentiation of hMSCs in basal growth media without the addition of exogenous phosphate in a manner that can be severely reduced, but not eliminated, by knockdown of the sodium phosphate transporters PiT-1 or PiT-2. The contributions of PiT-1 and PiT-2 to MC-GAG-mediated osteogenesis are nonredundant but also nonadditive, suggestive that the heterodimeric form is essential to its activity. These findings indicate that the mineral content of MC-GAG alters phosphate concentrations within a local microenvironment resulting in osteogenic differentiation of progenitor cells via both PiT-1 and PiT-2.
Assuntos
Osteogênese , Fosfatos , Humanos , Fosfatos/farmacologia , Alicerces Teciduais , Colágeno , Diferenciação Celular , Glicosaminoglicanos , Células CultivadasRESUMO
BACKGROUND/OBJECTIVE: Online physician-rating websites are being increasingly used by patients to gauge physicians' quality of service. The objective of this study is to assess the impact of residency training background of hair restoration surgeons and to identify themes of patient reviews using online physician-rating portals. METHOD: From July 1, 2018 to July 18, 2018, a list of American Board of Hair Restoration Surgery (ABHRS) physicians was compiled. Each member's name was searched on Healthgrades.com and Yelp.com websites, and profile reviews were analyzed for themes of patient satisfaction and dissatisfaction. RESULTS: Eighty-six ABHRS-certified surgeons were identified on the ABHRS physician directory practicing in the United States. Eleven different residency training backgrounds are represented. Surgeons were given significantly higher ratings on Healthgrades (4.63 vs 3.77, P < 0.001), and nonsurgeons were given significantly higher ratings on Yelp (4.31 vs 4.11, P = 0.036). A total of 567 five-star reviews and 59 one-star reviews were analyzed for content across both physician review portals. The most commonly cited topics included results, office staff, physician's bedside manner, comfort during procedure, patient-perceived physician's honesty, patient-perceived physician's knowledge, cost/financing options, recovery time, and wait time/scheduling. CONCLUSIONS: Significant, yet inconsistent differences in ratings were found between surgical and nonsurgical residency backgrounds across online physician-rating portals. Understanding drivers of positive and negative reviews may help surgeons improve patient satisfaction.
Assuntos
Alopecia/cirurgia , Competência Clínica/estatística & dados numéricos , Internet/estatística & dados numéricos , Satisfação do Paciente/estatística & dados numéricos , Cirurgiões/estatística & dados numéricos , Feminino , Humanos , Masculino , Relações Médico-Paciente , Estados UnidosRESUMO
The ability of the extracellular matrix (ECM) to direct cell fate has generated the potential for developing a materials-only strategy for tissue regeneration. Previously, we described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material that efficiently induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without exogenous growth factors or progenitor cell expansion. In this work, we evaluated the interactions between MC-GAG and primary human osteoclasts (hOCs). In the absence of hMSCs, mineralized Col-GAG materials directly inhibited hOC viability, proliferation, and resorption in contrast to nonmineralized Col-GAG, which demonstrated a modest inhibition of resorptive activity only. Cocultures containing differentiating hMSCs with hOCs demonstrated increased hOC-mediated resorption only on Col-GAG while MC-GAG cocultures continued to inhibit resorption. Unlike Col-GAG, hMSCs on MC-GAG expressed increased amounts of osteoprotegerin (OPG) protein, the major endogenous osteoclast inhibitor. Interestingly, OPG expression was found to be antagonized by small mothers against decapentaplegic1/5 (Smad1/5) phosphorylation, an obligate pathway for osteogenic differentiation of hMSCs on MC-GAG, and potentiated by extracellular signal-regulated kinase (ERK1/2) phosphorylation. Collectively, these results suggested that the MC-GAG material both directly inhibited the osteoclast viability, proliferation, and resorptive activity as well as induced hMSCs to secrete osteoprotegerin, an antiosteoclastogenic factor, via a signalling pathway distinct from osteogenic differentiation.
Assuntos
Diferenciação Celular , Colágeno/química , Glicosaminoglicanos/química , Células-Tronco Mesenquimais/metabolismo , Nanopartículas/química , Osteoclastos/metabolismo , Proliferação de Células , Sobrevivência Celular , Humanos , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais/citologia , Osteoclastos/citologiaRESUMO
The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.
Assuntos
Reabsorção Óssea/genética , Calcificação Fisiológica/genética , Osteogênese/genética , Osteoprotegerina/genética , Alicerces Teciduais , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/genética , Reabsorção Óssea/prevenção & controle , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Técnicas de Cocultura , Colágeno Tipo I/química , Colágeno Tipo I/farmacologia , Reagentes de Ligações Cruzadas/química , Expressão Gênica , Glicosaminoglicanos/química , Glicosaminoglicanos/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Cultura Primária de Células , Engenharia Tecidual , TransgenesRESUMO
OBJECT Craniosynostosis is often treated with neurosurgical intervention. The aim of this study was to report and analyze the clinical and socioeconomic characteristics of patients with craniosynostosis and to present current national trends. METHODS Using the Kids' Inpatient Database for the years 2000, 2003, 2006, and 2009, the authors identified patients with craniosynostosis using International Classification of Diseases, Ninth Revision, Clinical Modification diagnosis codes and their associated procedure codes. Clinical features, demographics, inpatient procedures, outcomes, and charges were collected and analyzed. RESULTS Of the 3415 patients identified, 65.8% were White, 21.4% were Hispanic, and 3.2% were Black. More than 96% were treated at urban teaching hospitals and 54.2% in southern or western regions. White patients were younger (mean 6.1 months) as compared with Blacks (mean 10.9 months) and Hispanics (mean 9.1 months; p < 0.0001) at the time of surgery. A higher fraction of Whites had private insurance (70.3%) compared with nonwhites (34.0%-41.6%; p < 0.001). Approximately 12.2% were nonelective admissions, more so among Blacks (16.9%). Mean hospital length of stay (LOS) was 3.5 days with no significant differences among races. Following surgical treatment, 12.1% of patients developed complications, most commonly pulmonary/respiratory (4.8%), wound infection (4.4%), and hydrocephalus (1.4%). The mean overall hospital charges were significantly lower for Whites than nonwhites ($34,527 vs $44,890-$48,543, respectively; p < 0.0001). CONCLUSIONS The findings of this national study suggest a higher prevalence of craniosynostosis in Hispanics. The higher predisposition among males was less evident in Hispanics and Blacks. There was a significant percentage of nonelective admissions, more commonly among Blacks. Additionally, Hispanics and Blacks were more likely to receive surgery at an older age, past the current recommendation of the optimum age for surgical intervention. These findings are likely associated with a lack of early detection. Although mean LOS and rate of complications did not significantly differ among different races, nonwhites had, on average, higher hospital charges of $10,000-$14,000. This discrepancy may be due to differences in type of insurance, craniosynostosis type, rates of comorbidities, and delay in treatment. Although there are several limitations to this analysis, the study reports on relevant disparities regarding a costly neurosurgical intervention, and ways to diminish these disparities should be further explored.
Assuntos
População Negra/etnologia , Craniossinostoses , Hispânico ou Latino/etnologia , Preços Hospitalares/estatística & dados numéricos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Complicações Pós-Operatórias , Sistema de Registros/estatística & dados numéricos , População Branca/etnologia , Craniossinostoses/economia , Craniossinostoses/etnologia , Craniossinostoses/cirurgia , Feminino , Humanos , Lactente , Tempo de Internação/economia , Tempo de Internação/estatística & dados numéricos , Masculino , Complicações Pós-Operatórias/economia , Complicações Pós-Operatórias/etnologia , Fatores Socioeconômicos , Estados Unidos/etnologiaRESUMO
PURPOSE: PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study, we investigated whether PD-L1 expression is controlled by melanoma driver mutations and modified by oncogenic signaling inhibition. EXPERIMENTAL DESIGN: Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors (BRAFi). The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated. RESULTS: No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN, or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, whereas the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines, the effects of BRAF, MEK, and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1-exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines. CONCLUSIONS: In melanoma cell lines, including BRAFi-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1-exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies.
Assuntos
Antígeno B7-H1/genética , Melanoma/genética , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Indóis/farmacologia , Linfócitos/imunologia , Linfócitos/metabolismo , Melanoma/imunologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , VemurafenibRESUMO
Adenovirus-mediated delivery of the immune-stimulatory cytokine Flt3L and the conditionally cytotoxic thymidine kinase (TK) induces tumor regression and long-term survival in preclinical glioma (glioblastoma multiforme [GBM]) models. Flt3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. Although pDCs can present antigen and produce powerful inflammatory cytokines, that is, interferon α (IFN-α), their role in tumor immunology remains debated. Thus, we studied the role of pDCs and IFN-α in Ad.TK/GCV+ Ad.Flt3L-mediated anti-GBM therapeutic efficacy. Our data indicate that the combined gene therapy induced recruitment of plasmacytoid DCs (pDCs) into the tumor mass; which were capable of in vivo phagocytosis, IFN-α release, and T-cell priming. Thus, we next used either pDCs or an Ad vector encoding IFN-α delivered within the tumor microenvironment. When rats were treated with Ad.TK/GCV in combination with pDCs or Ad-IFN-α, they exhibited 35% and 50% survival, respectively. However, whereas intracranial administration of Ad.TK/GCV + Ad.Flt3L exhibited a high safety profile, Ad-IFN-α led to severe local inflammation, with neurologic and systemic adverse effects. To elucidate whether the efficacy of the immunotherapy was dependent on IFN-α-secreting pDCs, we administered an Ad vector encoding B18R, an IFN-α antagonist, which abrogated the antitumoral effect of Ad.TK/GCV + Ad.Flt3L. Our data suggest that IFN-α release by activated pDCs plays a critical role in the antitumor effect mediated by Ad.TK/GCV + Ad.Flt3L. In summary, taken together, our results demonstrate that pDCs mediate anti-GBM therapeutic efficacy through the production of IFN-α, thus manipulation of pDCs constitutes an attractive new therapeutic target for the treatment of GBM.
Assuntos
Neoplasias Encefálicas/imunologia , Células Dendríticas/imunologia , Glioblastoma/imunologia , Interferon-alfa/imunologia , Microambiente Tumoral , Adenoviridae/genética , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Terapia Genética , Glioblastoma/patologia , Glioblastoma/terapia , Imunoterapia , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Membrana/genética , Ratos , Linfócitos T/imunologia , Timidina Quinase/genéticaRESUMO
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. GBM is very aggressive due to its poor cellular differentiation and invasiveness, which makes complete surgical resection virtually impossible. Therefore, GBM's invasive nature as well as its intrinsic resistance to current treatment modalities makes it a unique therapeutic challenge. Extensive examination of human GBM specimens has uncovered that these tumors overexpress a variety of receptors that are virtually absent in the surrounding non-neoplastic brain. Human GBMs overexpress receptors for cytokines, growth factors, ephrins, urokinase-type plasminogen activator (uPA), and transferrin, which can be targeted with high specificity by linking their ligands with highly cytotoxic molecules, such as Diptheria toxin and Pseudomonas exotoxin A. We review the preclinical development and clinical translation of targeted toxins for GBM. In view of the clinical experience, we conclude that although these are very promising therapeutic modalities for GBM patients, efforts should be focused on improving the delivery systems utilized in order to achieve better distribution of the immuno-toxins in the tumor/resection cavity. Delivery of targeted toxins using viral vectors would also benefit enormously from improved strategies for local delivery.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Imunotoxinas/administração & dosagem , Imunotoxinas/metabolismo , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , HumanosRESUMO
The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of 15-18 months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted; this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors.
Assuntos
Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Glioma/terapia , Toxinas Biológicas/uso terapêutico , Animais , Neoplasias Encefálicas/genética , Terapia Combinada , Marcação de Genes , Glioma/genética , Humanos , Imunoterapia , Interferons/uso terapêutico , Interleucinas/uso terapêutico , Modelos BiológicosRESUMO
We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6(-/-) mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6(-/-) mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.
Assuntos
Linfócitos B/imunologia , Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Glioblastoma/terapia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos B/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Citotoxicidade Imunológica/imunologia , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Herpesvirus Humano 1/enzimologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Fator 1 de Ligação ao Domínio I Regulador Positivo , Linfócitos T/metabolismo , Timidina Quinase/genética , Timidina Quinase/imunologia , Timidina Quinase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: Glioblastoma multiforme is a deadly primary brain cancer. Because the tumor kills due to recurrences, we tested the hypothesis that a new treatment would lead to immunological memory in a rat model of recurrent glioblastoma multiforme. EXPERIMENTAL DESIGN: We developed a combined treatment using an adenovirus (Ad) expressing fms-like tyrosine kinase-3 ligand (Flt3L), which induces the infiltration of immune cells into the tumor microenvironment, and an Ad expressing herpes simplex virus-1-thymidine kinase (TK), which kills proliferating tumor cells in the presence of ganciclovir. RESULTS: This treatment induced immunological memory that led to rejection of a second glioblastoma multiforme implanted in the contralateral hemisphere and of an extracranial glioblastoma multiforme implanted intradermally. Rechallenged long-term survivors exhibited anti-glioblastoma multiforme-specific T cells and displayed specific delayed-type hypersensitivity. Using depleting antibodies, we showed that rejection of the second tumor was dependent on CD8(+) T cells. Circulating anti-glioma antibodies were observed when glioblastoma multiforme cells were implanted intradermally in naïve rats or in long-term survivors. However, rats bearing intracranial glioblastoma multiforme only exhibited circulating antitumoral antibodies upon treatment with Ad-Flt3L + Ad-TK. This combined treatment induced tumor regression and release of the chromatin-binding protein high mobility group box 1 in two further intracranial glioblastoma multiforme models, that is, Fisher rats bearing intracranial 9L and F98 glioblastoma multiforme cells. CONCLUSIONS: Treatment with Ad-Flt3L + Ad-TK triggered systemic anti-glioblastoma multiforme cellular and humoral immune responses, and anti-glioblastoma multiforme immunological memory. Release of the chromatin-binding protein high mobility group box 1 could be used as a noninvasive biomarker of therapeutic efficacy for glioblastoma multiforme. The robust treatment efficacy lends further support to its implementation in a phase I clinical trial.
Assuntos
Neoplasias Encefálicas/terapia , Citotoxicidade Imunológica/genética , Terapia Genética/métodos , Glioma/terapia , Imunidade Celular/genética , Imunidade Humoral/genética , Memória Imunológica/genética , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Genes Transgênicos Suicidas/genética , Genes Transgênicos Suicidas/fisiologia , Glioma/imunologia , Glioma/patologia , Humanos , Imunoterapia/métodos , Ativação Linfocitária/genética , Proteínas de Membrana/genética , Modelos Biológicos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Ratos , Indução de Remissão/métodos , Timidina Quinase/genética , Carga TumoralRESUMO
PURPOSE: In preparation for a phase I clinical trial using a combined cytotoxic/immunotherapeutic strategy with adenoviruses (Ad) expressing Flt3L (Ad-Flt3L) and thymidine kinase (Ad-TK) to treat glioblastoma (GBM), we tested the hypothesis that Ad-TK+GCV would be the optimal tumor-killing agent in relation to efficacy and safety when compared with other proapoptotic approaches. EXPERIMENTAL DESIGN: The efficacy and neurotoxicity of Ad-TK+GCV was compared with Ads encoding the proapoptotic cytokines [tumor necrosis factor-alpha, tumor necrosis factor-related apoptosis-inducing factor (TRAIL), and Fas ligand (FasL)], alone or in combination with Ad-Flt3L. In rats bearing small GBMs (day 4), only Ad-TK+GCV or Ad-FasL improved survival. RESULTS: In rats bearing large GBMs (day 9), the combination of Ad-Flt3L with Ad-FasL did not improve survival over FasL alone, whereas Ad-Flt3L combined with Ad-TK+GCV led to 70% long-term survival. Expression of FasL and TRAIL caused severe neuropathology, which was not encountered when we used Ad-TK+/-Ad-Flt3L. In vitro, all treatments elicited release of high mobility group box 1 protein (HMGB1) from dying tumor cells. In vivo, the highest levels of circulating HMGB1 were observed after treatment with Ad-TK+GCV+Ad-Flt3L; HMGB1 was necessary for the therapeutic efficacy of AdTK+GCV+Ad-Flt3L because its blockade with glycyrrhizin completely blocked tumor regression. We also showed the killing efficacy of Ad-TK+GCV in human GBM cell lines and GBM primary cultures, which also elicited release of HMGB1. CONCLUSIONS: Our results indicate that Ad-TK+GCV+Ad-Flt3L exhibit the highest efficacy and safety profile among the several proapoptotic approaches tested. The results reported further support the implementation of this combined approach in a phase I clinical trial for GBM.