RESUMEN
Preventing tumor recurrence after surgical resection of a brain tumor is a significant clinical challenge because current methods deliver chemotherapeutic agents in a rapid manner and are not effective against the residual tumor cells. To overcome this drawback, we report a simple method to prepare magnetic resonance imaging (MRI) traceable ultra-thermosensitive hydrogels with rapid gelation ability from aqueous solution within 4 s at 28 °C for hydrophilic (epirubicin, EPI) and hydrophobic (paclitaxel, PTX) drugs co-delivery with bovine serum albumin nanoparticles (BSA NPs) incorporation. The results showed the average survival of gliosarcoma-bearing (MBR 614 or U87) mice receiving BSA/PTX NPs incorporated hydrogelGd/EPI increased to 63 days or 69 days with no tumor recurrence observed. Our synergistic strategy presents a new approach to the development of a local drug delivery system for the prevention of brain tumor recurrence.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Epirrubicina/administración & dosificación , Gliosarcoma/tratamiento farmacológico , Hidrogeles , Bombas de Infusión Implantables , Paclitaxel/administración & dosificación , Animales , Neoplasias Encefálicas/prevención & control , Modelos Animales de Enfermedad , Quimioterapia Combinada/métodos , Gliosarcoma/prevención & control , Humanos , Imagen por Resonancia Magnética , Ratones , Neoplasia Residual/tratamiento farmacológico , Prevención Secundaria , Análisis de Supervivencia , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
BACKGROUND: Vaccination against tumour-associated antigens is one approach to elicit anti-tumour responses. We investigated the effect of polynucleotide (DNA) vaccination using a model antigen (E. coli lacZ) in a syngeneic gliosarcoma model (9L). METHODS: Fisher 344 rats were vaccinated thrice by intramuscular injection of a lacZ-encoding or a control plasmid in weekly intervals. One week after the last vaccination, lacZ-expressing 9L cells were implanted into the striatum. RESULTS: After 3 weeks, in lacZ-vaccinated animals the tumours were significantly smaller than in control-vaccinated animals. In cytotoxic T cell assays lysis rates of >50 % could only be observed in a few of the lacZ-vaccinated animals. This response was directed against lacZ-expressing and parental 9L cells but not against syngeneic MADB 106 adenocarcinoma cells. In Elispot assays interferon-γ production was observed upon stimulation with 9LlacZ and 9L wild-type but not MADB 106 cells. This response was higher for lacZ-immunized animals. All animals revealed dense infiltrates with CD8+ lymphocytes and, to a lesser extent, with NK cells. CD25-staining indicated cells possibly associated with the maintenance of peripheral tolerance to self-antigens. All tumours were densely infiltrated by microglia consisting mostly of ramified cells. Only focal accumulation of macrophage-like cells expressing ED1, a marker for phagocytic activity, was observed. CONCLUSION: Prophylactic DNA vaccination resulted in effective but incomplete suppression of brain tumour formation. Mechanisms other than cytotoxic T cell responses as measured in the generally used in vitro assays appear to play a role in tumour suppression.
Asunto(s)
Neoplasias Encefálicas/patología , Neoplasias Encefálicas/prevención & control , Vacunas contra el Cáncer , Gliosarcoma/patología , Gliosarcoma/prevención & control , Vacunas de ADN , Animales , Antígenos Bacterianos/inmunología , Modelos Animales de Enfermedad , Escherichia coli/inmunología , Masculino , Ratas , Ratas Endogámicas F344 , beta-Galactosidasa/inmunologíaRESUMEN
Sustained local delivery of single agents and controlled delivery of multiple chemotherapeutic agents are sought for the treatment of brain cancer. A resorbable, multi-reservoir polymer microchip drug delivery system has been tested against a tumor model. The microchip reservoirs were loaded with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU was more stable at 37 degrees C within the microchip compared to a uniformly impregnated polymeric wafer (70% intact drug vs. 38%, at 48 h). The half-life of the intact free drug in the microchip was 11 days, which is a marked enhancement compared to its half-life in normal saline and 10% ethanol (7 and 10 min, respectively) [P. Tepe, S.J. Hassenbusch, R. Benoit, J.H. Anderson, BCNU stability as a function of ethanol concentration and temperature, J. Neurooncol. 10 (1991) 121-127; P. Kari, W.R. McConnell, J.M. Finkel, D.L. Hill, Distribution of Bratton-Marshall-positive material in mice following intravenous injections of nitrosoureas, Cancer Chemother. Pharmacol. 4 (1980) 243-248]. A syngeneic Fischer 344 9L gliosarcoma rat model was used to study the tumoricidal efficacy of BCNU delivery from the microchip or homogeneous polymer wafer. A dose-dependent decrease in tumor size was found for 0.17, 0.67, and 1.24 mg BCNU-microchips. Tumors treated with 1.24 mg BCNU-microchips showed significant tumor reduction (p=0.001) compared to empty control microchips at two weeks. The treatment showed similar efficacy to a polymer wafer with the same dosage. The microchip reservoir array may enable delivery of multiple drugs with independent release kinetics and formulations.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Materiales Biocompatibles , Carmustina/farmacología , Portadores de Fármacos , Implantes de Medicamentos , Gliosarcoma/prevención & control , Nanotecnología/métodos , Polímeros/química , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/uso terapéutico , Carmustina/química , Carmustina/farmacocinética , Carmustina/uso terapéutico , Química Farmacéutica , Ácidos Decanoicos/química , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Estabilidad de Medicamentos , Femenino , Gliosarcoma/patología , Semivida , Ácido Láctico/química , Poliésteres/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas , Ratas Endogámicas F344 , Reproducibilidad de los Resultados , Solubilidad , Factores de TiempoRESUMEN
Immunotherapy of gliomas has been forwarded as an attractive alternative to standard therapeutic modalities. Numerous observations indicate some therapeutic efficacy with this approach, but it is not curative in most reports. It is well established that gliomas suppress immune reactivity via a number of mechanisms, including expression CD95 ligand (CD95L), which induces apoptosis of immune effector cells, and secretion of immunosuppressive factors such as transforming growth factor-beta (TGFbeta). It has been hypothesized that abrogation of production or function of TGFbeta would improve immune reactivity to gliomas. To investigate this in a fashion that is translatable into clinical practice, we utilized a retroviral vector encoding a truncated, soluble form of the Type II receptor for TGFbeta (TFGbeta sr) and expressed it in the rat 9L gliosarcoma line (9L-TGFbeta sr). We then determined whether expression of TGFbeta sr affected in vitro sensitivity of 9L to lysis by immune effector cells, whether expression of TGFbeta sr affected tumorigenesis of 9L in vivo, and whether TGFbeta sr affected expression of immunity to 9L. In these experiments, we determined that 9L-TGFbeta sr was more susceptible than sham transfected 9L (9L-neo) to lysis by natural killer (NK) cells. We also determined that subcutaneously implanted 9L-TGFbeta sr was less tumorigenic than 9L-neo in syngeneic rats. Similarly, survival was extended by approximately 40% in rats given intracranial 9L-TGFbeta sr compared to 9L-neo. Finally, we determined that elimination of CD161+ cells resulted in comparable growth of 9L-neo and 9L-TGFbeta sr in vivo, indicating that NK or NK-like cells were responsible for the anti-tumor effects in this model.
Asunto(s)
Citotoxicidad Inmunológica , Gliosarcoma/metabolismo , Gliosarcoma/patología , Células Asesinas Naturales/inmunología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/inmunología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Gliosarcoma/inmunología , Gliosarcoma/prevención & control , Células Asesinas Naturales/efectos de los fármacos , Lectinas Tipo C/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK , Ratas , Ratas Endogámicas F344 , Receptores de Factores de Crecimiento Transformadores beta/química , Neoplasias Cutáneas/patología , Solubilidad , Análisis de Supervivencia , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta2RESUMEN
OBJECTIVE: On the basis of recent studies indicating that tumoral apoptotic bodies may provide a potent source of antigen for delivery to antigen-presenting cells, as well as observations that signal transduction modulation may constitute a promising approach for inducing glioma cell apoptosis, we explored the efficacy of vaccination with glioma apoptotic body-pulsed dendritic cells (DCs) for inhibiting tumor growth in the syngeneic 9L glioma/Fischer rat model. METHODS: For induction of apoptosis, 7-hydroxystaurosporine (UCN-01) (200-300 ng/ml), a selective protein kinase C inhibitor, was co-incubated with 9L cells in vitro for 72 or 96 hours. After this pretreatment period, glioma cells and DCs were mixed, and the interaction between DCs and apoptotic 9L tumor cells was assessed using two-color flow cytometry. In a series of experiments, the efficacy of vaccination strategies using DCs co-cultured with apoptotic 9L cells was then examined in animals harboring intracranial tumors. RESULTS: Pretreatment of 9L cells with UCN-01 resulted in approximately 50% of cells' being observed to undergo apoptosis as compared with less than 3% of controls. After subsequent co-culture, two-color flow cytometry demonstrated a time-dependent physical association of DCs with the apoptotic glioma cells. Survival in animals harboring intracranial tumors was significantly longer for the animals treated with a glioma apoptotic body-pulsed DC vaccine than in the animals that received apoptotic glioma cells and DCs alone or vehicle (i.e., the controls), especially those that underwent a sequential vaccination strategy (P < 0.0001). Long-term survival (>90 d) was demonstrated in 6 (75%) of 8 animals that underwent this vaccination approach versus 0 (0%) of 16 controls. In contrast, no survival benefit was observed in animals that received DCs that were co-cultured with vehicle-treated (non-apoptotic) 9L cells. Three of four long-term survivors that were rechallenged intracranially with tumor cells also survived over the long term. CONCLUSION: These studies suggest that induction of apoptosis in glioma cells by use of UCN-01 may promote the uptake of tumor antigens by DCs. This finding is important because apoptotic body-stimulated DCs may hold promise in promoting a host response against an established intracranial glioma, particularly if the parameters for apoptotic induction, duration of co-culture, and vaccination can be optimized.
Asunto(s)
Alcaloides/farmacología , Antígenos de Neoplasias/inmunología , Apoptosis , Neoplasias Encefálicas/prevención & control , Neoplasias Encefálicas/fisiopatología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Inhibidores Enzimáticos/farmacología , Glioma/prevención & control , Glioma/fisiopatología , Animales , Neoplasias Encefálicas/patología , Vacunas contra el Cáncer/administración & dosificación , Comunicación Celular , Células Dendríticas/fisiología , Esquema de Medicación , Glioma/patología , Gliosarcoma/prevención & control , Ratas , Estaurosporina/análogos & derivados , Células Tumorales Cultivadas , VacunaciónRESUMEN
Tumors arising within the central nervous system (CNS) present the immune system with a challenging target, given the heterogeneous nature of these neoplasms and their location within an "immunologically privileged" site. We used the lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) as a pseudotumor antigen to investigate recombinant Listeria monocytogenes as a tumor vaccine against s.c. and intracerebral challenges with a NP-expressing glioma, 9L-NP. Using Fischer 344 rats, we demonstrate that vaccination with recombinant L. monocytogenes-NP stimulates protection against s.c., but not intracerebral, 9L-NP tumor challenge in an antigen-specific, CD8(+) T-cell-dependent manner. After s.c. tumor rejection, enhanced antitumor immunity is achieved via epitope spreading that permits complete resistance against lethal intracerebral challenge with 9L-NP and with the untransfected parental 9L tumor. Unlike the CD8(+)-dependent immune responses against s.c. 9L-NP tumors, this expanded intracerebral immunity against endogenous tumor-associated antigens is dependent on both CD4(+) and CD8(+) T cells. Taken together, these results demonstrate that the mechanisms of tumor immunity within the brain are different from those elicited against non-CNS tumors. Furthermore, vaccination approaches exploiting the concept of epitope spreading may enhance the efficacy of antitumor immune responses within the immunologically privileged CNS, potentially mediating tumor cell killing through both CD4(+)- and CD8(+)-dependent effector pathways.
Asunto(s)
Neoplasias Encefálicas/inmunología , Vacunas contra el Cáncer/inmunología , Gliosarcoma/inmunología , Listeria monocytogenes/inmunología , Animales , Antígenos Virales/inmunología , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/prevención & control , Neoplasias Encefálicas/terapia , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Femenino , Gliosarcoma/patología , Gliosarcoma/prevención & control , Gliosarcoma/terapia , Activación de Linfocitos/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Nucleoproteínas/inmunología , Ratas , Ratas Endogámicas F344 , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Glioblastoma multiforme (GBM) is the most common primary human brain tumor. About 7000 new cases are diagnosed yearly in the USA. Despite current neurosurgical and postoperative radiotherapeutic tumor cytoreduction methods, in most cases occult foci of tumor cells infiltrate surrounding edematous brain tissues and cause recurrent disease within one year. GBM is almost invariably fatal within a few years after it is diagnosed. Our goal is to achieve long-term control of GBM by combining immunoprophylaxis with a radiation-based technique, such as boron neutron-capture therapy (BNCT), potentially capable of specifically targeting the infiltrating tumor cells while sparing the surrounding normal brain tissue. It has long been known that the subcutaneous (sc) injection of irradiated cells or untreated cultured cells (and the removal of the resulting tumors) derived from the well characterized, highly immunogenic 9L gliosarcoma (9LGS) rat model into young isogenic rats can prevent tumor growth after subsequent sc or intracranial (ic) injection of untreated, otherwise lethal 9LGS cells. In this study we have confirmed, quantified and extended those findings to study the efficacy of such immunological memory in normal aging rats and in aging rats previously treated for ic 9LGS tumors by BNCT. (1) The sc injection of 5,000,000 untreated 9LGS cells and the surgical removal of the resulting tumors (method A) protected 80% of normal young rats from an ic challenge with 10,000 untreated 9LGS cells, and a single sc injection of 5,000,000 lethally X-irradiated 9LGS cells (method B) protected 66% of them, but multiple sc injections with a crude particulate fraction prepared from 9LGS cells were not protective. Protection is long-lasting since contralateral ic rechallenge of six-month survivors with an injection of 10,000 viable 9LGS cells resulted in 100% survival. (2) Normal one-year-old rats were only slightly less protected than were normal young rats, approximately 70% rather than approximately 80% (method A) and approximately 60% rather than approximately 66% (method B). (3) BNCT treatment alone resulted in partial immunological protection, as 30% of one-year post-BNCT survivors of ic 9LGS tumors prevailed after contralateral ic rechallenge with 10,000 viable 9LGS cells. Moreover a single sc immunization with 5,000,000 untreated 9LGS cells prior to ic rechallenge boosted survival from 30% to 100%. The relevance of these observations to strategies of preclinical experimentation for immunoprophylaxis of malignant gliomas is discussed.
Asunto(s)
Terapia por Captura de Neutrón de Boro , Neoplasias Encefálicas/terapia , Vacunas contra el Cáncer/uso terapéutico , Gliosarcoma/terapia , Memoria Inmunológica , Inmunoterapia Activa , Vacunación , Animales , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/prevención & control , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/cirugía , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Terapia Combinada , Gliosarcoma/inmunología , Gliosarcoma/prevención & control , Gliosarcoma/radioterapia , Gliosarcoma/cirugía , Inmunidad Innata , Inyecciones Subcutáneas , Masculino , Trasplante de Neoplasias/inmunología , Ratas , Ratas Endogámicas F344 , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/efectos de la radiación , Células Tumorales Cultivadas/trasplanteRESUMEN
OBJECT: There have been numerous attempts to establish an effective immunotherapy for the treatment of brain tumors. To date, reliable methods to manipulate the immune system for promoting brain tumor regression have been disappointing. Generation of active immune responses in most of these studies was only possible in the absence of viable tumor cells, suggesting that immunotherapy can only be used as preventive therapy. In few studies the investigators have demonstrated success in using immunotherapy to treat a preestablished intracranial tumor. Using the 9L intracranial glioma model, the authors sought to delineate the underlying mechanisms for these observations. METHODS: In animals vaccinated with irradiated 9L glioma cells and interferon-gamma 14 and 7 days prior to intracranial tumor cell challenge, a significant increase in survival was shown. In contrast, vaccinations applied 3 days prior to, at the time of (Day 0) or 7 days after intracranial tumor cell challenge failed to influence survival. Histological examination of brain tissue specimens obtained in animals vaccinated before or after tumor cell challenge showed no difference in the degree of peritumoral mononuclear cell infiltration. When activated spleen cells obtained obtained from these animals were assayed for cytotoxicity and proliferative capacity, only those spleen cells derived from animals vaccinated prior to intracranial tumor cell challenge showed enhanced activity. CONCLUSIONS: These data support the presence of a strong modulatory effect of tumor on local and systemic antitumoral immune response. This immunosuppression appears to be secondary to a direct effect on T-cell function. Reversal of this immunosuppression may be a useful adjunct to tumor vaccine therapy.
Asunto(s)
Neoplasias Encefálicas/terapia , Vacunas contra el Cáncer , Gliosarcoma/terapia , Terapia de Inmunosupresión/métodos , Inmunoterapia/métodos , Animales , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/prevención & control , División Celular/inmunología , Línea Celular Tumoral/trasplante , Gliosarcoma/inmunología , Gliosarcoma/prevención & control , Interferón gamma/farmacología , Trasplante de Neoplasias , Neuroinmunomodulación/inmunología , Ratas , Ratas Endogámicas F344 , Tasa de Supervivencia , Linfocitos T Citotóxicos/inmunología , Factores de TiempoRESUMEN
Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3' phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5 x 10(5) or 2 x 10(6) irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized and non-immunized animals were challenged with various doses of intradermal (5 x 10(6)-5 x 10(7) or intracerebral (2 x 10(4)-5 x 10(5) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5 x 10(5) 9L-WT cells. With higher inocula up to 10(7) cells, tumors appeared in all the animals, but subsequently regressed in the immunized animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5 x 10(7) cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.
Asunto(s)
Neoplasias Encefálicas/inmunología , Quimiocina CCL2/uso terapéutico , Gliosarcoma/inmunología , Gliosarcoma/prevención & control , Neoplasias Cutáneas/prevención & control , Vacunas Sintéticas , Animales , Quimiocina CCL2/genética , Ingeniería Genética , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/uso terapéutico , Piel/inmunología , Transfección , Células Tumorales Cultivadas , Vacunas Sintéticas/inmunologíaRESUMEN
Phenylacetate is a naturally occurring plasma component that suppresses the growth of tumor cells and induces differentiation in vitro. To evaluate the in vivo potential and preventive and therapeutic antitumor efficacy of sodium phenylacetate against malignant brain tumors, Fischer 344 rats (n = 50) bearing cerebral 9L gliosarcomas received phenylacetate by continuous s.c. release starting on the day of tumor inoculation (n = 10) using s.c. osmotic minipumps (550 mg/kg/day for 28 days). Rats with established brain tumors (n = 12) received continuous s.c. phenylacetate supplemented with additional daily i.p. dose (300 mg/kg). Control rats (n = 25) were treated in a similar way with saline. Rats were sacrificed during treatment for electron microscopic studies of their tumors, in vivo proliferation assays, and measurement of phenylacetate levels in the serum and cerebrospinal fluid. Treatment with phenylacetate extended survival when started on the day of tumor inoculation (P < 0.01) or 7 days after inoculation (P < 0.03) without any associated adverse effects. In the latter group, phenylacetate levels in pooled serum and cerebrospinal fluid samples after 7 days of treatment were in the therapeutic range as determined in vitro (2.45 mM in serum and 3.1 mM in cerebrospinal fluid). Electron microscopy of treated tumors demonstrated marked hypertrophy and organization of the rough endoplasmic reticulum, indicating cell differentiation, in contrast to the scant and randomly distributed endoplasmic reticulum in tumors from untreated animals. In addition, in vitro studies demonstrated dose-dependent inhibition of the rate of tumor proliferation and restoration of anchorage dependency, a marker of phenotypic reversion. Phenylacetate, used at clinically achievable concentrations, prolongs survival of rats with malignant brain tumors through induction of tumor differentiation. Its role in the treatment of brain tumors and other cancers should be explored further.