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1.
Clin Exp Immunol ; 200(1): 33-44, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31784984

RESUMO

Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)-ß-mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single-cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways.


Assuntos
Neoplasias Encefálicas/imunologia , Perfilação da Expressão Gênica/métodos , Glioblastoma/imunologia , Tolerância Imunológica/imunologia , Células Matadoras Naturais/imunologia , Células-Tronco Neoplásicas/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Células Cultivadas , Estudos de Coortes , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Redes Reguladoras de Genes/imunologia , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Tolerância Imunológica/genética , Células Matadoras Naturais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Prognóstico , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
2.
Gene Ther ; 23(4): 357-68, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26814609

RESUMO

Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus (VV-NIS), GLV-1h153, was tested in in vitro analyzes of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumor radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and iodizing radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models showed that the addition of radioiodide to VV-NIS-infected tumors was more effective than each single-agent therapy, restricting tumor growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice.


Assuntos
Terapia Genética/métodos , Terapia Viral Oncolítica/métodos , Neoplasias da Próstata/terapia , Simportadores/genética , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Vírus Oncolíticos/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/virologia , Distribuição Aleatória , Simportadores/metabolismo , Transfecção , Vaccinia virus/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Clin Exp Immunol ; 180(1): 98-107, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25469725

RESUMO

Human natural killer (NK) cells play an important role in anti-viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon-inducible molecules CD69 and tetherin peaked 24-48 h post-infection, coincident with a peak of interferon-induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post-infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus-based therapy. Furthermore, our results suggest the existence of a post-infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.


Assuntos
Imunidade Celular , Células Matadoras Naturais/imunologia , Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Reoviridae/imunologia , Idoso , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Feminino , Humanos , Interferons/imunologia , Células Matadoras Naturais/patologia , Lectinas Tipo C/imunologia , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias/terapia
4.
Int J Cancer ; 134(5): 1091-101, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23982804

RESUMO

Reovirus is an oncolytic virus (OV), which acts by both direct tumor cell killing and priming of antitumor immunity. A major obstacle for effective oncolytic virotherapy is effective delivery of OV to tumor cells. Ovarian cancer is often confined to the peritoneal cavity and therefore i.p. delivery of reovirus may provide the ideal locoregional delivery, avoiding systemic dissemination. However, ovarian cancer is associated with an accumulation of ascitic fluid, which may interfere with oncolytic viral therapy. Here, we investigated the effect of ascites on reovirus-induced oncolysis against primary ovarian cancer cells and ovarian cancer cell lines. In the absence of ascites, reovirus was cytotoxic against ovarian cancer cells; however, cytotoxicity was abrogated in the presence of ascitic fluid. Neutralizing antibodies (NAb) were identified as the cause of this inhibition. Loading OV onto cell carriers may facilitate virus delivery in the presence of NAb and immune cells which have their own antitumor effector activity are particularly appealing. Immature dendritic cells (iDC), Lymphokine-activated killer (LAK) cells and LAKDC cocultures were tested as potential carriers for reovirus for tumor cell killing and immune cell priming. Reovirus-loaded LAKDC, and to a lesser degree iDC, were able to: (i) protect from NAb and hand-off reovirus for tumor cell killing; (ii) induce a proinflammatory cytokine milieu (IFNÉ£, IL-12, IFNα and TNFα) and (iii) generate an innate and specific antitumor adaptive immune response. Hence, LAKDC pulsed with reovirus represent a novel, clinically practical treatment for ovarian cancer to maximise both direct and innate/adaptive immune-mediated tumor cell killing.


Assuntos
Anticorpos Neutralizantes/imunologia , Ascite/imunologia , Células Dendríticas/imunologia , Células Matadoras Ativadas por Linfocina/imunologia , Terapia Viral Oncolítica , Neoplasias Ovarianas/terapia , Reoviridae/imunologia , Apoptose , Citocinas/biossíntese , Feminino , Humanos , Neoplasias Ovarianas/imunologia , Células Tumorais Cultivadas
5.
Surgeon ; 12(4): 210-20, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24502935

RESUMO

BACKGROUND: Despite mankind's many achievements, we are yet to find a cure for cancer. We are now approaching a new era which recognises the promise of harnessing the immune system for anti-cancer therapy. Pathogens have been implicated for decades as potential anti-cancer agents, but implementation into clinical therapy has been plagued with significant drawbacks. Newer 'designer' agents have addressed some of these concerns, in particular, a new breed of oncolytic virus: JX-594, a genetically engineered pox virus, is showing promise. OBJECTIVE: To review the current literature on the use of oncolytic viruses in the treatment of cancer; both by direct oncolysis and stimulation of the immune system. The review will provide a background and historical progression for the surgeon on tumour immunology, and the interplay between oncolytic viruses, immune cells, inflammation on tumourigenesis. METHODS: A literature review was performed using the Medline database. CONCLUSIONS: Viral therapeutics hold promise as a novel treatment modality for the treatment of disseminated malignancy. It provides a multi-pronged attack against tumour burden; direct tumour cell lysis, exposure of tumour-associated antigens (TAA), induction of immune danger signals, and recognition by immune effector cells.


Assuntos
Vacinas Anticâncer/uso terapêutico , Imunidade Celular , Neoplasias/terapia , Vírus Oncolíticos/imunologia , Vacinação/métodos , Humanos , Neoplasias/imunologia
6.
Gene Ther ; 20(1): 7-15, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22170342

RESUMO

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host antitumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.


Assuntos
Vírus do Sarampo/imunologia , Melanoma/imunologia , Vírus Oncolíticos/imunologia , Morte Celular/imunologia , Linhagem Celular Tumoral , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Vírus do Sarampo/patogenicidade , Melanoma/patologia , Melanoma/virologia , Vírus Oncolíticos/patogenicidade , Regulação para Cima
7.
Gene Ther ; 20(5): 521-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22895509

RESUMO

Oncolytic reovirus is currently under active investigation in a range of tumour types. Early phase studies have shown that this agent has modest monotherapy efficacy and its future development is likely to focus on combination regimens with cytotoxic chemotherapy. Indeed, phase I/II clinical trials have confirmed that reovirus can be safely combined with cytotoxic drugs, including a platin-taxane doublet regimen, which is currently being tested in a phase III clinical trial in patients with relapsed/metastatic head and neck cancer. Therefore, we have tested this triple (reovirus, cisplatin, paclitaxel) combination therapy in a panel of four head and neck cancer cell lines. Using the combination index (CI) method, the triple therapy demonstrated synergistic cytotoxicity in vitro in both malignant and non-malignant cell lines. In head and neck cancer cell lines, this was associated with enhanced caspase 3 and 7 cleavage, but no increase in viral replication. In vitro analyses confirmed colocalisation of markers of reovirus infection and caspase 3. Triple therapy was significantly more effective than reovirus or cisplatin-paclitaxel in athymic nude mice. These data suggest that the combination of reovirus plus platin-taxane doublet chemotherapy has significant activity in head and neck cancer and underpin the current phase III study in this indication.


Assuntos
Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Animais , Antineoplásicos , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Ensaios Clínicos Fase I como Assunto , Terapia Combinada , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Camundongos , Orthoreovirus/genética , Paclitaxel/administração & dosagem
8.
Br J Cancer ; 106(1): 92-8, 2012 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-22134504

RESUMO

BACKGROUND: Tumour cell lysates are an excellent source of many defined and undefined tumour antigens and have been used clinically in immunotherapeutic regimes but with limited success. METHODS: We conjugated Mel888 melanoma lysates to rabbit haemorrhagic disease virus virus-like particles (VLP), which can act as vehicles to deliver multiple tumour epitopes to dendritic cells (DC) to effectively activate antitumour responses. RESULTS: Virus-like particles did not stimulate the phenotypic maturation of DC although, the conjugation of lysates to VLP (VLP-lysate) did overcome lysate-induced suppression of DC activation. Lysate-conjugated VLP enhanced delivery of antigenic proteins to DC, while the co-delivery of VLP-lysates with OK432 resulted in cross-priming of naïve T cells, with expansion of a MART1(+) population of CD8(+) T cells and generation of a specific cytotoxic response against Mel888 tumour cell targets. The responses generated with VLP-lysate and OK432 were superior to those stimulated by unconjugated lysate with OK432. CONCLUSION: Collectively, these results show that the combination of VLP-lysate with OK432 delivered to DC overcomes the suppressive effects of lysates, and enables priming of naïve T cells with superior ability to specifically kill their target tumour cells.


Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Vírion/imunologia , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Eletroforese em Gel de Poliacrilamida , Humanos , Frações Subcelulares
9.
Br J Cancer ; 106(3): 496-507, 2012 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-22240799

RESUMO

BACKGROUND: There are still no effective treatments for superficial bladder cancer (SBC)/non-muscle invasive bladder cancer. Following treatment, 20% of patients still develop metastatic disease. Superficial bladder cancer is often multifocal, has high recurrences after surgical resection and recurs after intravesical live Bacillus Calmette-Guérin. Oncovex(GALV/CD), an oncolytic herpes simplex virus-1, has shown enhanced local tumour control by combining oncolysis with the expression of a highly potent pro-drug activating gene and the fusogenic glycoprotein. METHODS: In vitro fusion/prodrug/apoptotic cell-based assays. In vivo orthotopic bladder tumour model, visualised by computed microtomography. RESULTS: Treatment of seven human bladder carcinoma cell lines with the virus resulted in tumour cell killing through oncolysis, pro-drug activation and glycoprotein fusion. Oncovex(GALV/CD) and mitomycin C showed a synergistic effect, whereas the co-administration with cisplatin or gemcitabine showed an antagonistic effect in vitro. Transitional cell cancer (TCC) cells follow an apoptotic cell death pathway after infection with Oncovex(GALV/CD) with or without 5-FC. In vivo results showed that intravesical treatment with Oncovex(GALV/CD) + prodrug (5-FC) reduced the average tumour volume by over 95% compared with controls. DISCUSSION: Our in vitro and in vivo results indicate that Oncovex(GALV/CD) can improve local tumour control within the bladder, and potentially alter its natural history.


Assuntos
Carcinoma de Células de Transição/terapia , Glicoproteínas/uso terapêutico , Recidiva Local de Neoplasia/terapia , Terapia Viral Oncolítica , Pró-Fármacos/uso terapêutico , Neoplasias da Bexiga Urinária/terapia , Administração Intravesical , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Cricetinae , Modelos Animais de Doenças , Feminino , Fluoruracila/farmacologia , Glicoproteínas/farmacologia , Herpesvirus Humano 1/genética , Humanos , Vírus da Leucemia do Macaco Gibão/genética , Recidiva Local de Neoplasia/patologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacologia , Ratos , Ratos Endogâmicos F344 , Neoplasias da Bexiga Urinária/patologia
10.
Oncoimmunology ; 11(1): 2066050, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35558159

RESUMO

Systemic relapse after radiotherapy and surgery is the major cause of disease-related mortality in sarcoma patients. Combining radiotherapy and immunotherapy is under investigation as a means to improve response rates. However, the immune contexture of sarcoma is understudied. Here, we use a retrospective cohort of sarcoma patients, treated with neoadjuvant radiotherapy, and TCGA data. We explore therapeutic targets of relevance to sarcoma, using genomics and multispectral immunohistochemistry to provide insights into the tumor immune microenvironment across sarcoma subtypes. Differential gene expression between radioresponsive myxoid liposarcoma (MLPS) and more radioresistant undifferentiated pleomorphic sarcoma (UPS) indicated UPS contained higher transcript levels of a number of immunotherapy targets (CD73/NT5E, CD39/ENTPD1, CD25/IL2RA, and 4-1BB/TNFRSF9). We focused on 4-1BB/TNFRSF9 and other costimulatory molecules. In TCGA data, 4-1BB correlated to an inflamed and exhausted phenotype. OX40/TNFRSF4 and 4-1BB/TNFRSF9 were highly expressed in sarcoma subtypes versus other cancers. Despite OX40 and 4-1BB being described as Treg markers, we identified that they delineate distinct tumor immune profiles. This was true for sarcoma and other cancers. While only a limited number of samples could be analyzed, spatial analysis of OX40 expression identified two diverse phenotypes of OX40+ Tregs, one associated with and one independent of tertiary lymphoid structures (TLSs). Patient stratification is of intense interest for immunotherapies. We provide data supporting the viewpoint that a cohort of sarcoma patients, appropriately selected, are promising candidates for immunotherapies. Spatial profiling of OX40+ Tregs, in relation to TLSs, could be an additional metric to improve future patient stratification.


Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Adulto , Humanos , Recidiva Local de Neoplasia , Estudos Retrospectivos , Sarcoma/genética , Sarcoma/terapia , Linfócitos T Reguladores , Microambiente Tumoral
11.
Br J Cancer ; 105(6): 787-95, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21847125

RESUMO

BACKGROUND: Optimal cellular immunotherapy for cancer should ideally harness both the innate and adaptive arms of the immune response. Lymphokine-activated killer cells (LAKs) can trigger early innate killing of tumour targets, whereas long-term adaptive-specific tumour control requires priming of CD8+ cytotoxic lymphocytes (CTLs) following acquisition of tumour-associated antigens (TAAs) by antigen-presenting cells such as dendritic cells (DCs). As DCs stimulate both innate and adaptive effectors, combination cell therapy using LAKs and DCs has the potential to maximise anti-tumour immune priming. METHODS: Reciprocal activation between human clinical grade LAKs and DCs on co-culture, and its immune consequences, was monitored by cell phenotype, cytokine release and priming of both innate and adaptive cytotoxicity against melanoma targets. RESULTS: Co-culture of DCs and LAKs led to phenotypic activation of natural killer (NK) cells within the LAK population, which was associated with increased production of inflammatory cytokines and enhanced innate cytotoxicity against tumour cell targets. The LAKs reciprocally matured DCs, and the combination of LAKs and DCs, on addition of melanoma cells, supported priming of specific anti-tumour CTLs better than DCs alone. CONCLUSION: Clinical-grade LAKs/DCs represents a practical, effective combination cell immunotherapy for stimulation of both innate and adaptive anti-tumour immunity in cancer patients.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Dendríticas/imunologia , Imunoterapia/métodos , Células Matadoras Ativadas por Linfocina/imunologia , Melanoma/terapia , Imunidade Adaptativa , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/biossíntese , Citotoxicidade Imunológica , Humanos , Imunidade Inata
12.
Gene Ther ; 16(5): 689-99, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19282847

RESUMO

Reovirus is a naturally occurring oncolytic virus currently in early clinical trials. However, the rapid induction of neutralizing antibodies represents a major obstacle to successful systemic delivery. This study addresses, for the first time, the ability of cellular carriers in the form of T cells and dendritic cells (DC) to protect reovirus from systemic neutralization. In addition, the ability of these cellular carriers to manipulate the subsequent balance of anti-viral versus anti-tumour immune response is explored. Reovirus, either neat or loaded onto DC or T cells, was delivered intravenously into reovirus-naive or reovirus-immune C57Bl/6 mice bearing lymph node B16tk melanoma metastases. Three and 10 days after treatment, reovirus delivery, carrier cell trafficking, metastatic clearance and priming of anti-tumour/anti-viral immunity were assessed. In naive mice, reovirus delivered either neat or through cell carriage was detectable in the tumour-draining lymph nodes 3 days after treatment, though complete clearance of metastases was only obtained when the virus was delivered on T cells or mature DC (mDC); neat reovirus or loaded immature DC (iDC) gave only partial early tumour clearance. Furthermore, only T cells carrying reovirus generated anti-tumour immune responses and long-term tumour clearance; reovirus-loaded DC, in contrast, generated only an anti-viral immune response. In reovirus-immune mice, however, the results were different. Neat reovirus was completely ineffective as a therapy, whereas mDC--though not iDC--as well as T cells, effectively delivered reovirus to melanoma in vivo for therapy and anti-tumour immune priming. Moreover, mDC were more effective than T cells over a range of viral loads. These data show that systemically administered neat reovirus is not optimal for therapy, and that DC may be an appropriate vehicle for carriage of significant levels of reovirus to tumours. The pre-existing immune status against the virus is critical in determining the balance between anti-viral and anti-tumour immunity elicited when reovirus is delivered by cell carriage, and the viral dose and mode of delivery, as well as the immune status of patients, may profoundly affect the success of any clinical anti-tumour viral therapy. These findings are therefore of direct translational relevance for the future design of clinical trials.


Assuntos
Células Dendríticas/transplante , Melanoma Experimental/secundário , Melanoma Experimental/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/imunologia , Linfócitos T/transplante , Imunidade Adaptativa , Animais , Morte Celular , Citotoxicidade Imunológica , Linfonodos/virologia , Metástase Linfática , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reoviridae/imunologia , Reoviridae/isolamento & purificação , Resultado do Tratamento , Células Tumorais Cultivadas , Carga Viral
13.
Gene Ther ; 16(1): 78-92, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18784747

RESUMO

Transfer of healthy autologous tissue as a microvascular free flap facilitates reconstruction during ablative cancer surgery. In addition to filling surgical defects, free flaps might concentrate viral vectors at the tumour bed and mediate local therapeutic effects. We evaluated the magnitude, topography and duration of luciferase gene expression after plasmid and adenoviral delivery in rat superficial inferior epigastric (SIE) flaps. For plasmid delivery, luciferase expression was significantly increased by all transduction routes (topical, intraflap injection, intravascular) (P<0.01) at day 1, but not at day 7. The spread of luciferase expression was significantly different between the 4 groups at 1 day (P=0.026) and was greatest for flaps transduced by intravascular injection. For adenoviral transduction, total radiance was significantly different between the transduced groups at 1, 14 and 28 days (P<0.05 for all comparisons). The highest levels of radiance were seen in the intravascular group. There was a statistically significant difference in the spread of light emission between the 3 groups at 1 (P=0.009) and 14 (P=0.013) days, but this was no longer evident at 28 days. Intravascular adenoviral delivery yields high-level, diffuse and durable gene expression in rat SIE flaps and is suitable for examination in therapeutic models.


Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Plasmídeos/farmacologia , Retalhos Cirúrgicos , Animais , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Injeções , Óperon Lac , Luciferases/análise , Luciferases/genética , Masculino , Modelos Animais , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Transdução Genética/métodos
14.
Gene Ther ; 15(6): 424-33, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18079753

RESUMO

Escherichia coli nitroreductase (NTR) converts the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent that causes DNA crosslinks. In this study, the ability of NTR to enhance the combined effects of CB1954 and radiation has been tested in vitro and in vivo. Stably transduced ovarian cancer cells (SKOV3-NTR) that are sensitive to CB1954 (IC(50)=0.35 muM) demonstrated enhanced cytotoxicity when treated with CB1954 and single-fraction irradiation. The NTR-CB1954 system mediated a bystander effect in combination with radiation on transfer of conditioned medium from SKOV3-NTR, but not SKOV3, cells to SW480 target cells. The ability of CB1954 to enhance radiation-induced cytotoxicity in SKOV3-NTR (but not SKOV3) cells was also demonstrated by fluorescence-activated cell sorting (FACS) with dual staining for propidium iodide/fluorescein diacetate, 4',6-diamidino-2-phenylindole dichloride staining of apoptotic cells and measurement of double-stranded DNA breaks by FACS and confocal microscopy for gammaH2AX foci. Adenoviral delivery of NTR, under constitutive cytomegalovirus or tissue-specific CTP1 promoters, increased the in vitro cytotoxicity of CB1954 plus radiation in MTT and clonogenic assays. Finally, adenoviral delivery of NTR plus CB1954 enhanced the effect of fractionated radiotherapy (12 Gy in four fractions) in SW480 xenograft tumours in nude mice.


Assuntos
Aziridinas/administração & dosagem , Escherichia coli/enzimologia , Terapia Genética/métodos , Nitrorredutases/genética , Neoplasias Ovarianas/radioterapia , Radiossensibilizantes/administração & dosagem , Animais , Apoptose , Linhagem Celular Tumoral , Terapia Combinada , Citomegalovirus/genética , Fragmentação do DNA , Feminino , Citometria de Fluxo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Transdução Genética/métodos
15.
Gene Ther ; 15(18): 1257-70, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18401435

RESUMO

Reovirus is a promising unmodified double-stranded RNA (dsRNA) anti-cancer oncolytic virus, which is thought to specifically target cells with activated Ras. Although reovirus has been tested in a wide range of preclinical models and has entered early clinical trials, it has not previously been tested for the treatment of human melanoma. Here, we show that reovirus effectively kills and replicates in both human melanoma cell lines and freshly resected tumour; intratumoural injection also causes regression of melanoma in a xenograft in vivo model. Reovirus-induced melanoma death is blocked by caspase inhibition and is dependent on constituents of the Ras/RalGEF/p38 pathway. Reovirus melanoma killing is more potent than, and distinct from, chemotherapy or radiotherapy-induced cell death; a range of inflammatory cytokines and chemokines are released by infected tumour cells, while IL-10 secretion is abrogated. Furthermore, the inflammatory response generated by reovirus-infected tumour cells causes bystander toxicity against reovirus-resistant tumour cells and activates human myeloid dendritic cells (DC) in vitro. Hence, reovirus is suitable for clinical testing in melanoma, and may provide a useful danger signal to reverse the immunologically suppressive environment characteristic of this tumour.


Assuntos
Melanoma/terapia , Terapia Viral Oncolítica/métodos , Reoviridae/fisiologia , Neoplasias Cutâneas/terapia , Animais , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Cromonas/farmacologia , Citocinas/imunologia , Testes Imunológicos de Citotoxicidade , Células Dendríticas/imunologia , Citometria de Fluxo , Humanos , Imidazóis/farmacologia , Melanoma/imunologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/farmacologia , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/imunologia , Células Tumorais Cultivadas , Replicação Viral , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas ras/metabolismo
16.
Gene Ther ; 15(12): 911-20, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18323793

RESUMO

There is an emerging realization from animal models that the immune response may have both detrimental and beneficial therapeutic effects during cancer virotherapy. However, there is a dearth of clinical data on the immune response to viral agents in patients. During a recently completed phase I trial of intravenous reovirus type 3 Dearing (RT3D), heavily pretreated patients with advanced cancers received RT3D at doses escalating from 1 x 10(8) tissue culture infectious dose-50 (TCID(50)) on day 1 to 3 x 10(10) TCID(50) on 5 consecutive days of a 4 weekly cycle. A detailed analysis of the immune effects was conducted by collecting serial clinical samples for analysis of neutralizing anti-reoviral antibodies (NARA), peripheral blood mononuclear cells (PBMC) and cytokines. Significant increases in NARA were seen with peak endpoint titres >1/10 000 in all but one patient. The median fold increase was 250, with a range of 9-6437. PBMC subset analysis showed marked heterogeneity. At baseline, CD3+CD4+ T cells were reduced in most patients, but after RT3D therapy their numbers increased in 47.6% of patients. In contrast, most patients had high baseline CD3+CD8+ T-cell levels, with 33% showing incremental increases after therapy. In some patients, there was increased cytotoxic T-cell activation post-therapy, as shown by increased CD8+perforin/granzyme+ T-cell numbers. Most patients had high numbers of circulating CD3-CD56+ NK cells before therapy and in 28.6% this increased with treatment. Regulatory (CD3+CD4+CD25+) T cells were largely unaffected by the therapy. Combined Th1 and Th2 cytokine expression increased in 38% of patients. These data confirm that even heavily pretreated patients are capable of mounting dynamic immune responses during treatment with RT3D, although these responses are not clearly related to the administered virus dose. These data will provide the basis for future studies aiming to modulate the immune response during virotherapy.


Assuntos
Terapia Genética/métodos , Orthoreovirus Mamífero 3/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Citocinas/sangue , Feminino , Humanos , Imunidade Inata , Injeções Intravenosas , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Resultado do Tratamento
17.
Clin Oncol (R Coll Radiol) ; 20(2): 101-12, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18037277

RESUMO

The ability of the immune system to effectively respond to human tumours is a matter of long-term controversy. There is an increasing body of recent evidence to support a role for the immune system in eliminating pre-clinical cancers, an old concept termed 'immunosurveillance'. 'Immunoediting' is an updated hypothesis, in which selection pressures applied by the immune response to tumours modulate tumour immunogenicity and growth. Tumour infiltration by immune cells has been shown to have powerful prognostic significance in a host of cancer types. Paradoxically, in some circumstances the immune system can promote tumour development. Cytotoxic therapies, including radiotherapy and chemotherapy, induce potentially immunogenic cell death, releasing tumour-associated antigens in the context of a 'danger' signal to the immune system. An understanding of the interaction between immune cells, tumour cells and treatment modalities will therefore guide the future combination of immunotherapy with conventional therapy to achieve optimal anti-tumour effects.


Assuntos
Antineoplásicos/efeitos adversos , Sistema Imunitário/fisiologia , Neoplasias/imunologia , Animais , Antígenos de Neoplasias/fisiologia , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/efeitos da radiação , Tolerância Imunológica , Vigilância Imunológica , Camundongos , Modelos Imunológicos , Regressão Neoplásica Espontânea , Radioterapia/efeitos adversos , Evasão Tumoral
18.
Cancer Res ; 61(19): 7240-7, 2001 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11585761

RESUMO

The mechanisms by which the immune system distinguishes normal developmental cell death from pathological immunogenic cell killing are central to effective cancer immunotherapy. Using HSVtk suicide gene therapy, we showed that macrophages can distinguish between tumor cells dying through classical apoptosis and tumor cells engineered to die through nonapoptotic mechanisms, resulting in secretion of either immunosuppressive cytokines (interleukin 10 and transforming growth factor beta) or inflammatory cytokines (tumor necrosis factor alpha or interleukin 1beta), respectively. Additionally heat shock protein 70 acts as one component of a bimodal alarm signal that activates macrophages in the presence of stressful, immunogenic tumor cell killing. These differential responses of macrophages can also be used to vaccinate mice against tumor challenge, using adoptive transfer, as well as to cure mice of established tumors.


Assuntos
Apoptose/imunologia , Macrófagos/imunologia , Neoplasias Experimentais/imunologia , Animais , Vacinas Anticâncer/imunologia , Morte Celular/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Citocinas/imunologia , Citocinas/metabolismo , Ganciclovir/farmacologia , Terapia Genética , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/farmacologia , Imunoterapia Adotiva , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transfecção
20.
Leukemia ; 29(9): 1799-810, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25814029

RESUMO

The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Orthoreovirus Mamífero 3/imunologia , Vírus Oncolíticos/imunologia , Rituximab/imunologia , Rituximab/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Feminino , Humanos , Imunidade Inata , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Imunofenotipagem , Imunoterapia , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/diagnóstico , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Replicação Viral
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