RESUMEN
BACKGROUND: Reovirus exploits aberrant signalling downstream of Ras to mediate tumor-specific oncolysis. Since ~90% squamous cell carcinomas of the head and neck (SCCHN) over-express EGFR and SCCHN cell lines are sensitive to oncolytic reovirus, we conducted a detailed analysis of the effects of reovirus in 15 head and neck cancer cell lines. Both pre- and post-entry events were studied in an attempt to define biomarkers predictive of sensitivity/resistance to reovirus. In particular, we analysed the role of EGFR/Ras signalling in determining virus-mediated cytotoxicity in SCCHN. METHODS: To test whether EGFR pathway activity was predictive of increased sensitivity to reovirus, correlative analyses between reoviral IC50 by MTT assay and EGFR levels by western blot and FACS were conducted. Inhibition or stimulation of EGFR signalling were analysed for their effect on reoviral oncolysis by MTT assay, and viral growth by TCID50 assay. We next analysed the effects of inhibiting signalling downstream of Ras, by specific inhibitors of p38MAPK, PI3-K or MEK, on reoviral killing examined by MTT assay. The role of PKR in reoviral killing was also determined by blockade of PKR using 2-aminopurine and assaying for cell survival by MTT assay. The apoptotic response of SCCHN to reovirus was examined by western blot analysis of caspase 3 cleavage. RESULTS: Correlative analyses between reoviral sensitivity and EGFR levels revealed no association. Intermediate sub-viral and core particles showed the same infectivity/cytotoxicity as intact reovirus. Therefore, sensitivity was not determined by cell entry. In 4 cell lines, oncolysis and viral growth were both unaffected by inhibition or stimulation of EGFR signalling. Inhibition of signalling downstream of Ras did not abrogate reoviral oncolysis and, in addition, modulation of PKR using 2-aminopurine did not alter reovirus sensitivity in resistant cell lines. Caspase 3 cleavage was not detected in infected cells and oncolysis was observed in pan-caspase inhibited cells. CONCLUSIONS: In summary, reovirus is potently oncolytic in a broad panel of SCCHN cell lines. Attempts to define sensitivity/resistance by analysis of the EGFR/Ras/MAPK pathway have failed to provide a clear predictive biomarker of response. Further analysis of material from in vitro and clinical studies is ongoing in an attempt to shed further light on this issue.
Asunto(s)
Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/virología , Virus Oncolíticos/metabolismo , Infecciones por Reoviridae/metabolismo , Transducción de Señal/fisiología , Western Blotting , Línea Celular Tumoral , Receptores ErbB/metabolismo , Citometría de Flujo , Humanos , ReoviridaeRESUMEN
BACKGROUND: As well as inducing direct oncolysis, reovirus treatment of melanoma is associated with activation of innate and adaptive anti-tumour immune responses. RESULTS: Here we characterise the effects of conditioned media from reovirus-infected, dying human melanoma cells (reoTCM), in the absence of live virus, to address the immune bystander potential of reovirus therapy. In addition to RANTES, IL-8, MIP-1α and MIP-1ß, reovirus-infected melanoma cells secreted eotaxin, IP-10 and the type 1 interferon IFN-ß. To address the mechanisms responsible for the inflammatory composition of reoTCM, we show that IL-8 and IFN-ß secretion by reovirus-infected melanoma cells was associated with activation of NF-κB and decreased by pre-treatment with small molecule inhibitors of NF-κB and PKR; specific siRNA-mediated knockdown further confirmed a role for PKR. This pro-inflammatory milieu induced a chemotactic response in isolated natural killer (NK) cells, dendritic cells (DC) and anti-melanoma cytotoxic T cells (CTL). Following culture in reoTCM, NK cells upregulated CD69 expression and acquired greater lytic potential against tumour targets. Furthermore, melanoma cell-loaded DC cultured in reoTCM were more effective at priming adaptive anti-tumour immunity. CONCLUSIONS: These data demonstrate that the PKR- and NF-κB-dependent induction of pro-inflammatory molecules that accompanies reovirus-mediated killing can recruit and activate innate and adaptive effector cells, thus potentially altering the tumour microenvironment to support bystander immune-mediated therapy as well as direct viral oncolysis.
Asunto(s)
Quimiocinas/biosíntesis , Inmunidad/inmunología , Mediadores de Inflamación/metabolismo , Melanoma/inmunología , FN-kappa B/metabolismo , Reoviridae/inmunología , eIF-2 Quinasa/metabolismo , Inmunidad Adaptativa/efectos de los fármacos , Inmunidad Adaptativa/inmunología , Línea Celular Tumoral , Quimiocinas/metabolismo , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Inmunidad/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Melanoma/enzimología , Melanoma/metabolismo , Melanoma/virología , Fenotipo , Reoviridae/efectos de los fármacos , Infecciones por Reoviridae/inmunología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Oncolytic virotherapy may mediate antitumor effects via direct oncolysis or immune-mediated tumor regression. Although the ability of oncolytic viruses to generate adaptive antitumor immunity has been characterized, their interactions with the innate immune system are relatively unclear. Using a human in vitro system, this study investigates the innate immunological consequences of reovirus therapy and its potential to activate NK cell-mediated antitumor activity. Dendritic cells (DC) loaded with reovirus-infected human melanoma Mel888 cells (DC-MelReo), but not reovirus-infected tumor cells alone, induced IFN-gamma production within the NK cell population upon coculture with PBMC, in a cell-to-cell contact-dependent manner. DC-MelReo secreted the chemokines CCL2, 3, 4, 5, 7, 8, 11, and CXCL10; these culture supernatants induced NK cell chemotaxis. Coculture of DC-MelReo with purified NK cells induced reciprocal contact-dependent phenotypic DC maturation, while DC-MelReo elicited up-regulation of the activation marker CD69 on NK cells, in a partially contact and partially IL-12 dependent manner. Significantly, DC-MelReo induced NK cell cytotoxicity toward tumor cells by a type I IFN dependent mechanism. These data demonstrate that tumor infection by reovirus can act via DC to induce NK cell recruitment, activation, and cytotoxicity, along with reciprocal DC maturation. These findings suggest that reciprocal DC-NK cell interactions, following reovirus therapy, may play an important role in altering the immune milieu of the tumor microenvironment and mediating tumor regression.
Asunto(s)
Comunicación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Melanoma Experimental/inmunología , Melanoma Experimental/prevención & control , Reoviridae/inmunología , Línea Celular Tumoral , Células Cultivadas , Quimiotaxis de Leucocito/inmunología , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Células Dendríticas/patología , Humanos , Células Asesinas Naturales/patología , Melanoma Experimental/virología , Viroterapia Oncolítica/métodos , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/prevención & control , Infecciones por Reoviridae/virologíaRESUMEN
Fusion of tumor cells with antigen-presenting cells (APCs) has been proposed for the preparation of cancer vaccines. However, generation of these hybrids, using physical or chemical methods such as electrofusion or polyethylene glycol (PEG), has been difficult to standardize. Characterization of cell fusion has also been problematic because of difficulties in differentiating fusion from cell aggregation, leakage of cellular dyes and dendritic-cell (DC) phagocytosis of tumor material. In this report, we describe a new method to generate hybrid cell vaccines, based on gene transfer of a viral fusogenic membrane glycoprotein (FMG) into tumor cells, and incorporate a genetic method by which true hybrid formation can be unambiguously detected. We describe a new class of tumor cell-DC hybrid that can be rapidly isolated after cell fusion. These hybrids are highly potent in in vitro antigen presentation assays, target lymph nodes in vivo and are powerful immunogens against established metastatic disease.
Asunto(s)
Vacunas contra el Cáncer/genética , Células Dendríticas/citología , Técnicas Genéticas , Animales , Antígenos de Neoplasias , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Fusión Celular/métodos , Células Dendríticas/fisiología , Glicoproteínas/genética , Células Híbridas , Ganglios Linfáticos/patología , Melanoma/genética , Melanoma/patología , Melanoma/terapia , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Células Tumorales Cultivadas , VacunaciónRESUMEN
In vivo, dendritic cells (DC) are programmed to orchestrate innate and adaptive immunity in response to pathogen-derived "danger" signals. Under particular circumstances, DC can also be directly cytotoxic against tumor cells, potentially allowing them to release tumor associated Ags from dying cells and then prime antitumor immunity against them. In this study, we describe the innate characteristics of DC (OK-DC) generated in vitro after exposure of immature human myeloid-derived DC to OK432, a penicillin-inactivated and lyophilized preparation of Streptococcus pyrogenes. OK-DC produced proinflammatory cytokines, stimulated autologous T cell proliferation and IFN-gamma secretion, expressed CCR7, and migrated in response to MIP-3beta. Moreover, OK-DC displayed strong, specific cytotoxicity toward tumor cell targets. This cytotoxicity was associated with novel, OK432-induced up-regulation of CD40L on the cell surface of OK-DC, and was absolutely dependent on expression of CD40 on the tumor targets. These data demonstrate that maturation of human DC with OK432, an adjuvant suitable for clinical use, induces direct tumor cell killing by DC, and describes a novel CD40/CD40L-mediated mechanism for specific DC antitumor cytotoxicity.
Asunto(s)
Antígenos CD40/metabolismo , Ligando de CD40/metabolismo , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Neoplasias/inmunología , Picibanil/farmacología , Ligando de CD40/genética , Muerte Celular , Células Dendríticas/efectos de los fármacos , Humanos , Neoplasias/patología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
PURPOSE: To test combination treatment schedules of reovirus and radiation in human and murine tumor cells in vitro and in vivo. EXPERIMENTAL DESIGN: In vitro cytotoxicity and cell cycle effects of reovirus given alone and combined with radiotherapy were assessed by colorimetric, tissue culture infectious dose 50, and fluorescence-activated cell sorting-based assays. Interactions between the agents were evaluated using combination index analysis. The effect of different schedules of reovirus and radiotherapy on viral replication and cytotoxicity was tested in vitro and the combination was assessed in three tumor models in vivo. RESULTS: Characterization of reovirus cytotoxicity in a panel of cell lines yielded a range of sensitivities. Combined reovirus and radiotherapy yielded statistically significantly increased cytotoxicity, particularly in cell lines with moderate susceptibility to reovirus alone. The enhanced cytotoxicity of the combination occurred independently of treatment sequence or schedule. Radiation did not affect viral replication and only reduced reoviral cytotoxicity after clinically irrelevant single doses (>50 Gy). Combination index analysis revealed synergy between radiation (3-10 Gy) and reovirus at multiplicities of infection between 0.001 and 1. Combination treatment significantly increased apoptosis in tumor cells relative to either single-agent treatment. In vivo studies using xenograft and syngeneic tumors showed enhanced activity of the combination relative to reovirus or radiation alone (P < 0.001). CONCLUSIONS: Combining reovirus and radiotherapy synergistically enhances cytotoxicity in a variety of tumor cells in vitro and in vivo. These results offer strong support for translational clinical trials of reovirus plus radiotherapy that have been initiated in the clinic.
Asunto(s)
Supervivencia Celular/efectos de la radiación , Neoplasias/virología , Reoviridae/patogenicidad , Adulto , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Neoplasias Colorrectales/radioterapia , Neoplasias Colorrectales/virología , Terapia Combinada , Virus del Dengue , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Cabeza y Cuello/virología , Humanos , Rayos XRESUMEN
PURPOSE: Early clinical trials are under way exploring the direct oncolytic potential of reovirus. This study addresses whether tumor infection by reovirus is also able to generate bystander, adaptive antitumor immunity. EXPERIMENTAL DESIGN: Reovirus was delivered intravenously to C57BL/6 mice bearing lymph node metastases from the murine melanoma, B16-tk, with assessment of nodal metastatic clearance, priming of antitumor immunity against the tumor-associated antigen tyrosinase-related protein-2, and cytokine responses. In an in vitro human system, the effect of reovirus infection on the ability of Mel888 melanoma cells to activate and load dendritic cells for cytotoxic lymphocyte (CTL) priming was investigated. RESULTS: In the murine model, a single intravenous dose of reovirus reduced metastatic lymph node burden and induced antitumor immunity (splenocyte response to tyrosinase-related protein-2 and interleukin-12 production in disaggregated lymph nodes). In vitro human assays revealed that uninfected Mel888 cells failed to induce dendritic cell maturation or support priming of an anti-Mel888 CTL response. In contrast, reovirus-infected Mel888 cells (reo-Mel) matured dendritic cells in a reovirus dose-dependent manner. When cultured with autologous peripheral blood lymphocytes, dendritic cells loaded with reo-Mel induced lymphocyte expansion, IFN-gamma production, specific anti-Mel888 cell cytotoxicity, and cross-primed CD8+ T cells specific against the human tumor-associated antigen MART-1. CONCLUSION: Reovirus infection of tumor cells reduces metastatic disease burden and primes antitumor immunity. Future clinical trials should be designed to explore both direct cytotoxic and immunotherapeutic effects of reovirus.
Asunto(s)
Antineoplásicos/uso terapéutico , Citotoxicidad Inmunológica/inmunología , Neoplasias Experimentales/terapia , Viroterapia Oncolítica/métodos , Infecciones por Reoviridae/inmunología , Animales , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Interferón gamma/biosíntesis , Oxidorreductasas Intramoleculares/inmunología , Metástasis Linfática/patología , Activación de Linfocitos/inmunología , Antígeno MART-1 , Orthoreovirus Mamífero 3/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/virología , Reacción en Cadena de la Polimerasa , Linfocitos T Citotóxicos/inmunologíaRESUMEN
PURPOSE: Fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus G glycoprotein (VSV-G), represent a new class of gene therapy for cancer that cause cytotoxic fusion on expression in tumor cells. In addition, FMG-mediated tumor cell death stimulates antitumor immunity, suggesting potential applications for FMG-expressing cellular vaccines. This study addresses the promise of FMG-expressing allogeneic tumor cells, which are most practical for clinical use, as a novel platform for ex vivo and in situ vaccination. EXPERIMENTAL DESIGN: Murine B16 melanoma-derived cell lines expressing autologous or allogeneic MHC class I, expressing fusogenic or nonfusogenic VSV-G, were used to vaccinate mice in vivo against a live tumor challenge. Exosome-like vesicles released by fusing allogeneic cells (syncitiosomes) and intratumoral injection of fusing vaccines were also tested as novel therapeutic strategies for their antitumor effects. RESULTS: Expression of fusogenic VSV-G enhanced the immunogenicity of an allogeneic cellular vaccine, which was more effective than a fusing autologous vaccine. Allogeneic syncitiosomes were only as effective as cellular vaccines when administered with adjuvant, demonstrating that syncitiosomes cannot account entirely for the mechanism of immune priming. Intratumoral injection of FMG-expressing allogeneic cells led to significant tumor regression using both fusogenic or nonfusogenic VSV-G. However, specific priming against tumor-associated antigenic epitopes and protection against secondary rechallenge only occurred if the initial vaccine was competent for cell fusion. CONCLUSIONS: FMG-expressing allogeneic tumor cells are a potent source of antitumor vaccines. Syncitiosomes given with adjuvant and intratumoral injection of fusing cells represent novel strategies well-suited to clinical translation.
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Inmunoterapia Adoptiva/métodos , Melanoma Experimental/terapia , Glicoproteínas de Membrana/genética , Proteínas del Envoltorio Viral/genética , Animales , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Vesículas Citoplasmáticas/inmunología , Expresión Génica , Antígenos H-2/genética , Antígenos H-2/inmunología , Melanoma Experimental/genética , Melanoma Experimental/patología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Inducción de Remisión , Factores de Tiempo , Transfección , Trasplante Homólogo , Resultado del Tratamiento , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
We have investigated how to make K1735 cells, a poor allogeneic melanoma vaccine, more effective for protection against B16 in vivo. To promote antigen release in an immunologically effective manner, tumor cells were transfected with a viral fusogenic membrane glycoprotein (vesicular stomatitis virus G glycoprotein), which kills cells through the formation, and degeneration, of large multinucleated syncytia. Vaccines consisting of a 1:1 mix of fusing allogeneic and autologous cells led to dramatic increases in survival of mice in both prophylactic and therapy models, dependent upon T cells, the mechanism of tumor-tumor cell fusion, and the nature of the fusion partner. Syncytia activate macrophages and fusogenic membrane glycoprotein-mediated cell killing very efficiently promotes cross-priming of immature dendritic cells with a model tumor antigen. Our data suggest that the unique manner in which syncytia develop and die provides a highly effective pathway for tumor antigen release and presentation to the immune system and offers a novel mechanism by which cancer cell vaccines may be prepared for clinical use.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Fusión Celular/métodos , Melanoma Experimental/terapia , Glicoproteínas de Membrana/inmunología , Proteínas del Envoltorio Viral/inmunología , Células 3T3 , Animales , Presentación de Antígeno/inmunología , Antígenos de Neoplasias , Muerte Celular/inmunología , Técnicas de Cocultivo , Células Dendríticas/inmunología , Proteínas del Huevo/inmunología , Activación de Macrófagos/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Antígenos Específicos del Melanoma , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/inmunología , Ovalbúmina/inmunología , Fragmentos de PéptidosRESUMEN
Idarubicin (IDA) is an anthracycline used during treatment of acute myelogenous leukaemia (AML) and is clinically important because of its potency and lipophilicity (compared to the related compounds daunorubicin and doxorubicin). These drugs target DNA topoisomerase II (topo II), a nuclear enzyme that regulates DNA topology. Topo II poisoning leads to the trapping of an intermediate in the enzyme's cycle termed the "cleavable complex." This study aims to increase understanding of drug interactions by use of the "TARDIS" (trapped in agarose DNA immunostaining) assay to measure drug-induced topo II cleavable complexes in individual cells treated with anthracyclines. Mammalian cells contain two isoforms of topo II (alpha and beta) and the TARDIS assay enables visualisation of isoform-specific complexes. Drug-treated cells were embedded in agarose, lysed and incubated with anti-topo II antibodies to microscopically detect topo IIalpha or beta complexes. Results for K562 cells (at clinically relevant concentrations) showed that IDA and idarubicinol, its metabolite, formed mainly topo IIalpha cleavable complexes, the level of which decreases at doses > 1 microM for IDA. Our data suggest that this decrease is due to catalytic inhibition by IDA at these doses. Doxorubicin formed low levels of topo IIalpha complexes and negligible topo IIbeta complexes. In cytotoxicity studies, IDA and idarubicinol were equipotent, but both were more potent than daunorubicin and doxorubicin. We showed for the first time that there was a persistent increase in levels of topo IIalpha cleavable complexes after removal of IDA, suggesting that its greater effectiveness may be associated with both the longevity and high levels of these complexes.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Idarrubicina/farmacología , Dominio Catalítico/efectos de los fármacos , ADN/efectos de los fármacos , ADN/metabolismo , Daunorrubicina/farmacología , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Células K562 , Leucemia/patología , Inhibidores de Topoisomerasa IIRESUMEN
PURPOSE: DNA topoisomerase II (topo II) is an important cellular target for chemotherapeutic agents. Human cells have two isoforms of topo II (alpha and beta), and both are inhibited by the chemotherapeutic agents etoposide, amsacrine (mAMSA) and mitoxantrone. It is known that the cytotoxic importance of topo IIalpha or topo IIbeta drug-induced complexes differs depending on which drug is present. This study was designed to (a) assess isoform-specific formation and reversal of topo IIalpha and beta cleavable complexes, and (b) determine whether the cytotoxic importance of either isoform was related to differences in the longevity of the complexes. METHODS: Mouse embryonic fibroblasts (MEFs) were used to study the cellular response to the topo II poisons etoposide, mitoxantrone and mAMSA. The longevity of topo IIalpha and beta complexes was determined using the TARDIS assay. This immunofluorescence assay can differentiate between the topo II isoforms and thus allowed us to investigate the persistence and importance of topo IIalpha and beta complexes for the first time. RESULTS: In MEFs treated with etoposide, 50% of topo IIalpha complexes dissociated within 40 min whereas dissociation of topo IIbeta complexes took only 20 min. Disappearance of complexes was a slower process for mitoxantrone-treated cells. The time taken to reduce topo IIalpha and topo IIbeta cleavable complexes by 50% was 10 and 6 h, respectively. In contrast, mAMSA-stabilized topo IIalpha and topo IIbeta cleavable complexes were equally stable (dissociation within 15 min for both isoforms). These stability data were confirmed using an in vitro assay. CONCLUSIONS: We previously demonstrated that topo IIalpha is the major target for etoposide and mitoxantrone but that both topo IIalpha and topo IIbeta are important for mAMSA cytotoxicity. The longevity of the topo IIalpha and beta cleavable complexes shown here is therefore an important factor in determining the cytotoxic sensitivity of either isoform to these drugs.
Asunto(s)
Antineoplásicos/farmacología , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , Amsacrina/farmacología , Animales , Antígenos de Neoplasias , Antineoplásicos Fitogénicos/farmacología , ADN-Topoisomerasas de Tipo II/química , ADN de Neoplasias/química , ADN de Neoplasias/efectos de los fármacos , Proteínas de Unión al ADN , Etopósido/farmacología , Fibroblastos/enzimología , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Semivida , Isoenzimas/química , Isoenzimas/efectos de los fármacos , Cinética , Ratones , Mitoxantrona/farmacologíaRESUMEN
Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.
Asunto(s)
Antígenos de Neoplasias/genética , Vacunas contra el Cáncer/genética , Biblioteca de Genes , Neoplasias/terapia , Animales , Vacunas contra el Cáncer/inmunología , ADN Complementario/genética , Vectores Genéticos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/genética , Neoplasias/inmunología , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/terapia , Vesiculovirus/genéticaRESUMEN
PURPOSE: To test combination treatment schedules of reovirus and cisplatin chemotherapy in human and murine melanoma cell lines and murine models of melanoma and to investigate the possible mechanisms of synergistic antitumor effects. EXPERIMENTAL DESIGN: The effects of reovirus +/- chemotherapy on in vitro cytotoxicity and viral replication were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and plaque assay. Interactions between agents were assessed by combination index analysis. Mode of cell death was assessed by Annexin V/propidium iodide fluorescence-activated cell sorting-based assays; gene expression profiling of single versus combination treatments was completed using the Agilent microarray system. Single agent and combination therapy effects were tested in vivo in two immunocompetent models of murine melanoma. RESULTS: Variable degrees of synergistic cytotoxicity between live reovirus and several chemotherapy agents were observed in B16.F10 mouse melanoma cells, most significantly with cisplatin (combination index of 0.42 +/- 0.03 at ED(50)). Combination of cisplatin and reovirus exposure led to increased late apoptotic/necrotic cell populations. Cisplatin almost completely abrogated the inflammatory cytokine gene up-regulation induced by reovirus. Combination therapy led to significantly delayed tumor growth and improved survival in vivo (P < 0.0001 and P = 0.0003, respectively). Cisplatin had no effect on the humoral response to reovirus in mice. However, cisplatin treatment suppressed the cytokine and chemokine response to reovirus in vitro and in vivo. CONCLUSION: The combination of reovirus and several chemotherapeutic agents synergistically enhanced cytotoxicity in human and murine melanoma cell lines in vitro and murine tumors in vivo. The data support the current reovirus/chemotherapy combination phase I clinical studies currently ongoing in the clinic.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cisplatino/administración & dosificación , Melanoma Experimental/terapia , Viroterapia Oncolítica , Reoviridae/fisiología , Animales , Apoptosis/fisiología , Terapia Combinada , Humanos , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Virus Oncolíticos/fisiología , Resultado del Tratamiento , Células Tumorales Cultivadas , Replicación Viral/efectos de los fármacosRESUMEN
The three-way interaction between oncolytic viruses, the tumor microenvironment, and the immune system is critical to the outcome of antitumor therapy. Classically, the immune system is thought to limit the efficacy of therapy, leading to viral clearance. However, preclinical and clinical data suggest that in some cases virotherapy may in fact act as cancer immunotherapy. In this review we discuss the ability of oncolytic viruses to alter the immunogenic milieu of the tumor microenvironment, and the role of innate and adaptive immunity in both restricting and augmenting therapy. Strategies to improve virotherapy by immunomodulation, including suppression or enhancement of the innate and adaptive responses, are discussed.
Asunto(s)
Sistema Inmunológico/inmunología , Viroterapia Oncolítica , Virus Oncolíticos/inmunología , Animales , Ensayos Clínicos como Asunto , Humanos , Neoplasias/inmunología , Receptores de Reconocimiento de Patrones/inmunologíaRESUMEN
PURPOSE: Reovirus is a naturally occurring oncolytic virus in clinical trials. Although tumor infection by reovirus can generate adaptive antitumor immunity, its therapeutic importance versus direct viral oncolysis is undefined. This study addresses the requirement for viral oncolysis and replication, and the relative importance of antitumor immunity and direct oncolysis in therapy. EXPERIMENTAL DESIGN: Nonantigen specific T cells loaded with reovirus were delivered i.v. to C57BL/6 and severe combined immunodeficient mice bearing lymph node and splenic metastases from the murine melanoma, B16ova, with assessment of viral replication, metastatic clearance by tumor colony outgrowth, and immune priming. Human cytotoxic lymphocyte priming assays were done with dendritic cells loaded with Mel888 cells before the addition of reovirus. RESULTS: B16ova was resistant to direct oncolysis in vitro, and failed to support reovirus replication in vitro or in vivo. Nevertheless, reovirus purged lymph node and splenic metastases in C57BL/6 mice and generated antitumor immunity. In contrast, reovirus failed to reduce tumor burden in severe combined immunodeficient mice bearing either B16ova or reovirus-sensitive B16tk metastases. In the human system, reovirus acted solely as an adjuvant when added to dendritic cells already loaded with Mel888, supporting priming of specific antitumor cytotoxic lymphocyte, in the absence of significant direct tumor oncolysis; UV-treated nonreplicating reovirus was similarly immunogenic. CONCLUSION: The immune response is critical in mediating the efficacy of reovirus, and does not depend upon direct viral oncolysis or replication. The findings are of direct relevance to fulfilling the potential of this novel anticancer agent.
Asunto(s)
Citotoxicidad Inmunológica/fisiología , Orthoreovirus Mamífero 3/fisiología , Melanoma Experimental/terapia , Viroterapia Oncolítica , Virus Oncolíticos/fisiología , Replicación Viral/fisiología , Animales , Línea Celular Tumoral , Células Cultivadas , Humanos , Inmunoterapia Adoptiva/métodos , Orthoreovirus Mamífero 3/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/virología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Virus Oncolíticos/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/trasplante , Linfocitos T Citotóxicos/virología , Resultado del Tratamiento , Replicación Viral/inmunologíaRESUMEN
Oncolytic viruses are novel anticancer agents, currently under investigation in Phase I-III clinical trials. Until recently, most studies have focused on the direct antitumor properties of these viruses, although there is now an increasing body of evidence that the host immune response may be critical to the efficacy of oncolytic virotherapy. This may be mediated via innate immune effectors, adaptive antiviral immune responses eliminating infected cells or adaptive antitumor immune responses. This report summarizes preclinical and clinical evidence for the importance of immune interactions, which may be finely balanced between viral and tumor elimination. On this basis, oncolytic viruses represent a promising novel immunotherapy strategy, which may be optimally combined with existing therapeutic modalities.
Asunto(s)
Inmunoterapia , Neoplasias/terapia , Viroterapia Oncolítica , Adaptación Fisiológica , Animales , Ensayos Clínicos como Asunto , Terapia Combinada , Humanos , Neoplasias/inmunología , Neoplasias/patologíaRESUMEN
Oncolytic viruses are replication competent, tumor selective and lyse cancer cells. Their potential for anti-cancer therapy is based upon the concept that selective intratumoral replication will produce a potent anti-tumor effect and possibly bystander or remote cell killing, whilst minimizing normal tissue toxicity. Viruses may be naturally oncolytic or be engineered for oncolytic activity, and possess a host of different mechanisms to provide tumor selectivity. Clinical use of live replicating viruses is associated with a unique set of safety issues. Clinical experience has so far provided evidence of limited efficacy and a favourable toxicity profile. The interaction with the host immune system is complex. An anti-viral immune response may limit efficacy by rapidly clearing the virus. However, virally-induced cell lysis releases tumor associated antigens in a 'dangerous' context, and limited evidence suggests that this can lead to the generation of a specific anti-tumor immune response. Combination therapy with chemotherapy or radiotherapy represents a promising avenue for ongoing translation of oncolytic viruses into clinical practice. Obstacles to therapy include highly effective non-specific host mechanisms to clear virus following systemic delivery, immune-mediated clearance, and intratumoral barriers limiting virus spread. A number of novel strategies are now under investigation to overcome these barriers. This review provides an overview of the potential role of oncolytic viruses, highlighting recent progress towards developing effective therapy and asks if they are a realistic therapeutic option at this stage.
RESUMEN
Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-alpha, TNF-alpha, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-kappaB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-gamma production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-gamma) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus's direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.