RESUMEN

BACKGROUND: Temozolomide (TMZ) is an oral DNA-alkylating agent used for treating patients with glioblastoma. However, therapeutic benefits of TMZ can be compromised by the expression of O6-methylguanine methyltransferase (MGMT) in tumor tissue. Here we used MGMT-expressing glioblastoma stem cells (GSC) lines as a model for investigating the molecular mechanism underlying TMZ resistance, while aiming to explore a new treatment strategy designed to possibly overcome resistance to the clinically relevant dose of TMZ (35 µM). METHODS: MGMT-expressing GSC cultures are resistant to TMZ, and IC50 (half maximal inhibitory concentration) is estimated at around 500 µM. Clonogenic GSC surviving 500 µM TMZ (GSC-500 µM TMZ), were isolated. Molecular signatures were identified via comparative analysis of expression microarray against parental GSC (GSC-parental). The recombinant protein of top downregulated signature was used as a single agent or in combination with TMZ, for evaluating therapeutic effects of treatment of GSC. RESULTS: The molecular signatures characterized an activation of protective stress responses in GSC-500 µM TMZ, mainly including biotransformation/detoxification of xenobiotics, blocked endoplasmic reticulum stress-mediated apoptosis, epithelial-to-mesenchymal transition (EMT), and inhibited growth/differentiation. Bone morphogenetic protein 7 (BMP7) was identified as the top down-regulated gene in GSC-500 µM TMZ. Although augmenting BMP7 signaling in GSC by exogenous BMP7 treatment did not effectively stop GSC growth, it markedly sensitized both GSC-500 µM TMZ and GSC-parental to 35 µM TMZ treatment, leading to loss of self-renewal and migration capacity. BMP7 treatment induced senescence of GSC cultures and suppressed mRNA expression of CD133, MGMT, and ATP-binding cassette drug efflux transporters (ABCB1, ABCG2), as well as reconfigured transcriptional profiles in GSC by downregulating genes associated with EMT/migration/invasion, stemness, inflammation/immune response, and cell proliferation/tumorigenesis. BMP7 treatment significantly prolonged survival time of animals intracranially inoculated with GSC when compared to those untreated or treated with TMZ alone (p = 0.0017), whereas combination of two agents further extended animal survival compared to BMP7 alone (p = 0.0489). CONCLUSIONS: These data support the view that reduced endogenous BMP7 expression/signaling in GSC may contribute to maintained stemness, EMT, and chemoresistant phenotype, suggesting that BMP7 treatment may provide a novel strategy in combination with TMZ for an effective treatment of glioblastoma exhibiting unmethylated MGMT.

Asunto(s)

Proteína Morfogenética Ósea 7/metabolismo , Metilasas de Modificación del ADN/metabolismo , Dacarbazina/análogos & derivados , Glioblastoma/enzimología , Glioblastoma/metabolismo , Guanina/análogos & derivados , Células Madre Neoplásicas/enzimología , Antineoplásicos Alquilantes , Proteína Morfogenética Ósea 7/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Metilasas de Modificación del ADN/genética , Dacarbazina/farmacología , Resistencia a Antineoplásicos/genética , Glioblastoma/genética , Guanina/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Temozolomida , Células Tumorales Cultivadas

RESUMEN

Glioblastoma stem cells (GSC) co-exhibiting a tumor-initiating capacity and a radio-chemoresistant phenotype, are a compelling cell model for explaining tumor recurrence. We have previously characterized patient-derived, treatment-resistant GSC clones (TRGC) that survived radiochemotherapy. Compared to glucose-dependent, treatment-sensitive GSC clones (TSGC), TRGC exhibited reduced glucose dependence that favor the fatty acid oxidation pathway as their energy source. Using comparative genome-wide transcriptome analysis, a series of defense signatures associated with TRGC survival were identified and verified by siRNA-based gene knockdown experiments that led to loss of cell integrity. In this study, we investigate the prognostic value of defense signatures in glioblastoma (GBM) patients using gene expression analysis with Probeset Analyzer (131 GBM) and The Cancer Genome Atlas (TCGA) data, and protein expression with a tissue microarray (50 GBM), yielding the first TRGC-derived prognostic biomarkers for GBM patients. Ribosomal protein S11 (RPS11), RPS20, individually and together, consistently predicted poor survival of newly diagnosed primary GBM tumors when overexpressed at the RNA or protein level [RPS11: Hazard Ratio (HR) = 11.5, p<0.001; RPS20: HR = 4.5, p = 0.03; RPS11+RPS20: HR = 17.99, p = 0.001]. The prognostic significance of RPS11 and RPS20 was further supported by whole tissue section RPS11 immunostaining (27 GBM; HR = 4.05, p = 0.01) and TCGA gene expression data (578 primary GBM; RPS11: HR = 1.19, p = 0.06; RPS20: HR = 1.25, p = 0.02; RPS11+RPS20: HR = 1.43, p = 0.01). Moreover, tumors that exhibited unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) or wild-type isocitrate dehydrogenase 1 (IDH1) were associated with higher RPS11 expression levels [corr (IDH1, RPS11) = 0.64, p = 0.03); [corr (MGMT, RPS11) = 0.52, p = 0.04]. These data indicate that increased expression of RPS11 and RPS20 predicts shorter patient survival. The study also suggests that TRGC are clinically relevant cells that represent resistant tumorigenic clones from patient tumors and that their properties, at least in part, are reflected in poor-prognosis GBM. The screening of TRGC signatures may represent a novel alternative strategy for identifying new prognostic biomarkers.

Asunto(s)

Biomarcadores de Tumor/biosíntesis , Glioblastoma/genética , Proteínas Ribosómicas/biosíntesis , Adulto , Anciano , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Metilación de ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Pronóstico , Proteínas Ribosómicas/genética , Análisis de Matrices Tisulares

RESUMEN

Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment-introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD(+) levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma.

Asunto(s)

Adaptación Fisiológica/genética , Resistencia a Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glucosa/metabolismo , Células Madre/metabolismo , Estrés Fisiológico/genética , Línea Celular Tumoral , Reparación del ADN/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Expresión Génica/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/radioterapia , Humanos , Transducción de Señal/genética , Temozolomida , Regulación hacia Arriba/genética

RESUMEN

Glioblastoma stem cells (GSC) express both radial glial cell and neural crest cell (NCC)-associated genes. We report that endothelin 3 (EDN3), an essential mitogen for NCC development and migration, is highly produced by GSCs. Serum-induced proliferative differentiation rapidly decreased EDN3 production and downregulated the expression of stemness-associated genes, and reciprocally, two glioblastoma markers, EDN1 and YKL-40 transcripts, were induced. Correspondingly, patient glioblastoma tissues express low levels of EDN3 mRNA and high levels of EDN1 and YKL-40 mRNA. Blocking EDN3/EDN receptor B (EDNRB) signaling by an EDNRB antagonist (BQ788), or EDN3 RNA interference (siRNA), leads to cell apoptosis and functional impairment of tumor sphere formation and cell spreading/migration in culture and loss of tumorigenic capacity in animals. Using exogenous EDN3 as the sole mitogen in culture does not support GSC propagation, but it can rescue GSCs from undergoing cell apoptosis. Molecular analysis by gene expression profiling revealed that most genes downregulated by EDN3/EDNRB blockade were those involved in cytoskeleton organization, pause of growth and differentiation, and DNA damage response, implicating the involvement of EDN3/EDNRB signaling in maintaining GSC migration, undifferentiation, and survival. These data suggest that autocrine EDN3/EDNRB signaling is essential for maintaining GSCs. Incorporating END3/EDNRB-targeted therapies into conventional cancer treatments may have clinical implication for the prevention of tumor recurrence.

Asunto(s)

Comunicación Autocrina/genética , Neoplasias Encefálicas/genética , Endotelina-3/metabolismo , Glioblastoma/metabolismo , Cresta Neural/metabolismo , Receptor de Endotelina B/metabolismo , Animales , Apoptosis , Neoplasias Encefálicas/metabolismo , Diferenciación Celular , Movimiento Celular , Transformación Celular Neoplásica , Endotelina-3/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Genoma Humano , Glioblastoma/genética , Humanos , Ratones , Ratones SCID , Células Madre Neoplásicas , Cresta Neural/citología , Cresta Neural/crecimiento & desarrollo , ARN Interferente Pequeño , Receptor de Endotelina B/genética

RESUMEN

Glioblastoma (World Health Organization/WHO grade IV) is the most common and most aggressive adult glial tumor. Patients with glioblastoma, despite being treated with gross total resection and post-operative radiation/chemotherapy, will almost always develop tumor recurrence. Glioblastoma stem cells (GSC), a minor subpopulation within the tumor mass, have been recently characterized as tumor-initiating cells and hypothesized to be responsible for post-treatment recurrence because of their enhanced radio-/chemo-resistant phenotype and ability to reconstitute tumors in mouse brains. Genome-wide expression profile analysis uncovered molecular properties of GSC distinct from their differentiated, proliferative progeny that comprise the majority of the tumor mass. In contrast to the hyperproliferative and hyperangiogenic phenotype of glioblastoma tumors, GSC possess neuroectodermal properties and express genes associated with neural stem cells, radial glial cells, and neural crest cells, as well as portray a migratory, quiescent, and undifferentiated phenotype. Thus, cell cycle-targeted radio-chemotherapy, which aims to kill fast-growing tumor cells, may not completely eliminate glioblastoma tumors. To prevent tumor recurrence, a strategy targeting essential gene pathways of GSC must be identified and incorporated into the standard treatment regimen. Identifying intrinsic and extrinsic cues by which GSC maintain stemness properties and sustain both tumorigenesis and anti-apoptotic features may provide new insights into potentially curative strategies for treating brain cancers.

RESUMEN

Glioblastoma multiforme (GBM) remains refractory to conventional therapy. CD133+ GBM cells have been recently isolated and characterized as chemo-/radio-resistant tumor-initiating cells and are hypothesized to be responsible for post-treatment recurrence. In order to explore the molecular properties of tumorigenic CD133+ GBM cells that resist treatment, we isolated CD133+ GBM cells from tumors that are recurrent and have previously received chemo-/radio-therapy. We found that the purified CD133+ GBM cells sorted from the CD133+ GBM spheres express SOX2 and CD44 and are capable of clonal self-renewal and dividing to produce fast-growing CD133- progeny, which form the major cell population within GBM spheres. Intracranial injection of purified CD133+, not CD133- GBM daughter cells, can lead to the development of YKL-40+ infiltrating tumors that display hypervascularity and pseudopalisading necrosis-like features in mouse brain. The molecular profile of purified CD133+ GBM cells revealed characteristics of neuroectoderm-like cells, expressing both radial glial and neural crest cell developmental genes, and portraying a slow-growing, non-differentiated, polarized/migratory, astrogliogenic, and chondrogenic phenotype. These data suggest that at least a subset of treated and recurrent GBM tumors may be seeded by CD133+ GBM cells with neural and mesenchymal properties. The data also imply that CD133+ GBM cells may be clinically indolent/quiescent prior to undergoing proliferative cell division (PCD) to produce CD133- GBM effector progeny. Identifying intrinsic and extrinsic cues, which promote CD133+ GBM cell self-renewal and PCD to support ongoing tumor regeneration may highlight novel therapeutic strategies to greatly diminish the recurrence rate of GBM.

Asunto(s)

Antígenos CD/genética , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/patología , Glicoproteínas/genética , Células Madre Neoplásicas/fisiología , Péptidos/genética , Antígeno AC133 , Animales , Biomarcadores de Tumor/genética , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Femenino , Citometría de Flujo/métodos , Perfilación de la Expresión Génica/métodos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Células Madre Neoplásicas/citología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Esferoides Celulares/patología , Factores de Tiempo , Células Tumorales Cultivadas

RESUMEN

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent mediator of vascular permeability. VEGF inhibition reduces edema and tumor burden in some patients with malignant glioma, whereas others show no response. The role of VEGF expression in edema production and the relationship to survival is not well understood. EXPERIMENTAL DESIGN: Using DNA microarray analysis, we examined VEGF and related gene expression in 71 newly diagnosed malignant gliomas and analyzed the relationship to edema and survival. RESULTS AND CONCLUSIONS: VEGF expression was predictive of survival in tumors with little or no edema [Cox proportional hazard model, 6.88; 95% confidence interval (95% CI), 2.61-18.1; P<0.0001], but not in tumors with extensive edema. The expression of several proangiogenic genes, including adrenomedullin (correlation coefficient, 0.80), hypoxia-inducible factor-1A (0.51), and angiopoietin-2 (0.44), was correlated with VEGF expression (all with P<0.0001), whereas that of several antiangiogenic genes was inversely correlated. The expression of six genes was increased greater than 3-fold in edematous versus nonedematous tumors in the absence of increased VEGF expression. The most increased, neuronal pentraxin 2 (NPTX2, 7-fold change), was predictive of survival in tumors with the highest levels of edema, in contrast to VEGF (hazard ratio, 2.73; 95% CI, 1.49-5.02; P=0.049). NPTX2 was tightly correlated with expression of the water channel aquaporin-3 (0.74, P<0.0001). These results suggest that there are both VEGF-dependent and VEGF-independent pathways of edema production in gliomas and may explain why edema is not reduced in some patients following anti-VEGF treatment.

Asunto(s)

Edema Encefálico/diagnóstico , Neoplasias Encefálicas/mortalidad , Proteína C-Reactiva/genética , Glioma/mortalidad , Proteínas del Tejido Nervioso/genética , Factor A de Crecimiento Endotelial Vascular/genética , Adrenomedulina/genética , Angiopoyetina 2/genética , Acuaporina 3/genética , Edema Encefálico/genética , Neoplasias Encefálicas/complicaciones , Expresión Génica , Glioma/complicaciones , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Sobrevida

RESUMEN

Glioblastoma is the most common and aggressive primary brain cancer. Recent isolation and characterization of brain tumor-initiating cells supports the concept that transformed neural stem cells may seed glioblastoma. We previously identified a wide array of mesenchymal tissue transcripts overexpressed in a broad set of primary glioblastoma (de novo) tumors but not in secondary glioblastoma (derived from lower-grade) tumors, low-grade astrocytomas, or normal brain tissues. Here, we extend this observation and show that a subset of primary glioblastoma tumors and their derived tumor lines express cellular and molecular markers that are associated with mesenchymal stem cells (MSC) and that glioblastoma cell cultures can be induced to differentiate into multiple mesenchymal lineage-like cell types. These findings suggest either that a subset of primary glioblastomas derive from transformed stem cells containing MSC-like properties and retain partial phenotypic aspects of a MSC nature in tumors or that glioblastomas activate a series of genes that result in mesenchymal properties of the cancer cells to effect sustained tumor growth and malignant progression.

Asunto(s)

Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Mesenquimatosas/patología , Antígenos de Superficie/biosíntesis , Biopsia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Línea Celular Tumoral , Citometría de Flujo/métodos , Glioblastoma/genética , Glioblastoma/inmunología , Humanos , Inmunohistoquímica/métodos , Pérdida de Heterocigocidad/genética , Células Madre Mesenquimatosas/inmunología , Plasticidad Neuronal , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos

RESUMEN

Glioblastomas are invasive and aggressive tumors of the brain, generally considered to arise from glial cells. A subset of these cancers develops from lower-grade gliomas and can thus be clinically classified as "secondary," whereas some glioblastomas occur with no prior evidence of a lower-grade tumor and can be clinically classified as "primary." Substantial genetic differences between these groups of glioblastomas have been identified previously. We used large-scale expression analyses to identify glioblastoma-associated genes (GAG) that are associated with a more malignant phenotype via comparison with lower-grade astrocytomas. We have further defined gene expression differences that distinguish primary and secondary glioblastomas. GAGs distinct to primary or secondary tumors provided information on the heterogeneous properties and apparently distinct oncogenic mechanisms of these tumors. Secondary GAGs primarily include mitotic cell cycle components, suggesting the loss of function in prominent cell cycle regulators, whereas primary GAGs highlight genes typical of a stromal response, suggesting the importance of extracellular signaling. Immunohistochemical staining of glioblastoma tissue arrays confirmed expression differences. These data highlight that the development of gene pathway-targeted therapies may need to be specifically tailored to each subtype of glioblastoma.

Asunto(s)

Neoplasias Encefálicas/genética , Glioblastoma/genética , Glioblastoma/secundario , Adipoquinas , Apoptosis/genética , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Ciclo Celular/genética , Procesos de Crecimiento Celular/genética , Proteína 1 Similar a Quitinasa-3 , Perfilación de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/patología , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Humanos , Inmunohistoquímica , Lectinas , Mesodermo/patología , Células del Estroma/patología , Transcripción Genética , Regulación hacia Arriba

RESUMEN

MUC-1 is overexpressed on many tumor cells. In addition, aberrant glycosylation of MUC-1 on human tumors leads to exposure of cryptic peptide epitopes that play a role in tumor immunity. As such, it has been identified as a potential target for immunotherapy. The purpose of this phase 1 clinical trial was to determine the maximum tolerated dose, safety of a multiple-dose regimen, and the immunologic effect of vaccinia virus expressing MUC-1 and IL-2 genes (VV/MUC-1/IL-2) in patients with advanced prostate cancer. Five x 10(5), 5 x 10(6), and 5 x 10(7) plaque-forming units (pfu) of vaccinia viruses were used in the dose-escalating study. Viruses were given via intramuscular injection, and clinical response and immune function modulation were analyzed. No grade 3 or 4 toxicity was observed. Objective clinical response was observed after the fourth injection (0.3 ng/mL) in only one patient who received an intermediate dose of virus. Systemic immune modulation in this patient included (1) up-regulation of IL-2 (CD25) and T cell (TcR alphabeta) receptors, (2) increase in the CD4/CD8 ratio (2.5-fold) (3) augmentation of T-helper type 1 cell (TH1) (interferon-gamma and tumor necrosis factor-alpha) but not TH2 (IL-4) cytokine mRNA expression, (4) induction of natural killer cell activity and MHC independent MUC-1 specific cytotoxic T-cell activity, and (5) normalization of mRNA expression of T-cell-associated signal transduction molecules TcR-zeta and p56lck. These results suggest that VV/MUC-1/IL-2 gene therapy with a maximum tolerated dose of 5 x 10(7) pfu is safe and well tolerated.

Asunto(s)

Vacunas contra el Cáncer/uso terapéutico , Terapia Genética/métodos , Interleucina-2/genética , Mucina-1/genética , Neoplasias de la Próstata/terapia , Virus Vaccinia/genética , Anciano , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Separación Celular , Ensayos Clínicos como Asunto , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Vectores Genéticos , Humanos , Inmunoterapia , Interleucina-2/metabolismo , Masculino , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1 , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba

RESUMEN

Cultured tumor lysate-loaded dendritic cells (TuLy-DC) have been demonstrated in vitro to stimulate potent immune modulations and generate significant antitumor response. We report the results of a pilot trial of TuLy-DC vaccine for patients with metastatic renal cell carcinoma (mRCC). Fourteen mRCC patients underwent nephrectomy to obtain autologous TuLy prepared by subjecting tumor cells to 3 freeze/thaw cycles. Dendritic cells were generated from peripheral blood CD14+ precursors cultured in the presence of GM-CSF, IL-4, and 10% autologous serum. Patients received one vaccination of TuLy alone as an immunologic control, followed by 3 weekly vaccinations of DC-TuLy injected intradermally in the midaxillary region. Peripheral blood lymphocytes were collected before and after weekly vaccines and were assessed for changes in phenotype, cytotoxicity, and cytokine profile. The TuLy-DC vaccine was successfully prepared and administered to 12 patients, whereas 2 patients did not receive vaccine treatment due to declines in postoperative performance status. The vaccines were well tolerated, with only grade 1 toxicities noted. One patient had a partial response to treatment that did not correspond to any significant change in immunologic profile. This pilot trial demonstrated both the safety and feasibility of reliably preparing a DC-based vaccine for mRCC patients. Our data suggest that autologous TuLy-DC vaccines generate only limited clinical response. Further clinical studies are needed to identify the most potent treatment regimen that can consistently mediate an antitumor immune response in vivo.

Asunto(s)

Vacunas contra el Cáncer/uso terapéutico , Carcinoma de Células Renales/terapia , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Neoplasias Renales/terapia , Vacunación , Adulto , Anciano , Vacunas contra el Cáncer/efectos adversos , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/secundario , Carcinoma de Células Renales/cirugía , Células Cultivadas , Citocinas/metabolismo , Femenino , Humanos , Inmunofenotipificación , Neoplasias Renales/inmunología , Neoplasias Renales/cirugía , Masculino , Persona de Mediana Edad , Nefrectomía , Proyectos Piloto , Reacción en Cadena de la Polimerasa , Resultado del Tratamiento , Células Tumorales Cultivadas

RESUMEN

PURPOSE: We investigated the ability of the fusion protein granulocyte-macrophage colony-stimulating factor and carbonic anhydrase IX (GMCA-9)(1) to induce an immune response in vitro and in vivo for the development of a GMCA-9-based kidney cancer vaccine. EXPERIMENTAL DESIGN: Human dendritic cells (DCs) were transduced with a recombinant adenovirus containing the GMCA-9 gene and tested for their capacity to induce CA9-specific cytotoxic T lymphocytes in vitro. Tumor growth was studied in severe compromised immunodeficiency disease (SCID) mice s.c. injected with R11-GMCA-9, a human renal cell carcinoma cell line stably transfected with the GMCA-9 gene. Involvement of natural killer (NK) cells in the antitumor activity of GMCA-9 was determined in SCID mice treated with the NK-blocking agent anti-asialoGM-1. RESULTS: DC and R11 cells transduced with GMCA-9 produced a GMCA-9 protein that is targeted to the cell membrane and partially processed to granulocyte macrophage colony-stimulating factor- and CA9-like products. Furthermore, GMCA-9 was capable of inducing DC maturation, as well as CA9-specific cytotoxic lymphocytes in vitro. Tumor growth of R11 cells in SCID mice was significantly inhibited after transfection with the GMCA-9 fusion gene (P < 0.01). In mice treated with anti-asialoGM-1, R11-GMCA-9 tumors grew significantly faster than those of control mice (P < 0.05), suggesting an involvement of NK cells. CONCLUSIONS: Our results suggest that the fusion protein GMCA-9 is capable of generating an immune response both in vitro and in vivo. Additional studies will confirm the utility of ex vivo GMCA-9-transduced DCs as a kidney cancer vaccine.

Asunto(s)

Antígenos de Neoplasias/genética , Anhidrasas Carbónicas/genética , Carcinoma de Células Renales/terapia , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Inmunoterapia/métodos , Neoplasias Renales/terapia , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusión/farmacología , Adenoviridae/genética , Animales , Anhidrasa Carbónica IX , Carcinoma de Células Renales/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/fisiología , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Humanos , Isoenzimas , Neoplasias Renales/inmunología , Células Asesinas Naturales/inmunología , Masculino , Ratones , Ratones SCID , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/fisiología , Transducción Genética

RESUMEN

OBJECTIVE: We recently described a new hormone refractory prostate cancer cell line, CL1, derived from LNCaP via in vitro androgen deprivation. To study gene expression during prostate cancer progression and to identify molecular targets for therapy, a pure clonal tumor system was generated. METHODS: Limiting dilution of CL1 stably transfected with a green fluorescent protein, generated 35 single-cell clones, which were expanded into stable cell lines. In vitro responses to various therapeutic modalities were assessed in each clone. Gene expression was determined using reverse transcriptase-polymerase chain reaction and oligonucleotide microarrays. In vivo biology was assessed following orthotopic injection into intact and castrated severe combined immunodeficient mice. RESULTS: In vitro, all clones demonstrated similar resistance to traditional therapeutic efforts including chemotherapy and radiation therapy, but differential sensitivity to cell-mediated cytotoxicity. The clones demonstrated differential gene expression relative to each other and to the parental CL1 and LNCaP cell lines. Following orthotopic injection into mice, three distinct growth patterns were observed: fast growth with widespread metastasis; slower grower with widespread metastasis; and no tumor formation. Using oligonucleotide microarrays, several genes were identified as differentially expressed between the most aggressive and the nontumorigenic clone. CONCLUSIONS: We have described a novel fluorescent-labeled clonal hormone refractory prostate cancer tumor system that exhibited marked heterogeneity in its response to various therapeutic modalities, gene expression, and in vivo biology. Our data suggests that given the marked clonal heterogeneity, multi-modality approaches directed against multiple molecular targets rather than single agent therapy will be necessary to adequately eradicate the entire malignant cell population. Clonal tumor lines may allow more accurate examination of molecular pathways involved in tumor progression and resistance to treatment.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Animales , Células Clonales , Regulación Neoplásica de la Expresión Génica , Proteínas Fluorescentes Verdes , Histocitoquímica , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones SCID , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/patología , ARN Neoplásico/química , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

BACKGROUND: We hypothesized that the aggressive LNCaP-derived androgen-independent cell line, CL1, might differ from LNCaP in their repertoire of cell surface markers and that these differences might typify changes that occur during clinical prostate cancer progression. METHODS: The cell surface marker expression profiles of CL1 and LNCaP were examined using flow cytometry. Markedly differential gene expression was confirmed using RT-PCR and further examined using immunohistochemistry among the prostate cancer cell lines LAPC-4, LNCaP, CL1, CL2, DU145, and PC-3. The expression of the most markedly differentially expressed surface marker, CD10, was further explored in a tissue microarray containing radical prostatectomy samples from 219 hormone naïve prostate cancer patients. RESULTS: There were marked differences in the expression of CD10, CD13, CD26, CD33, CD44, CD54, CD55, and CD104 between CL1 and LNCaP. Results from both the RT-PCR and immunohistochemistry confirmed the differential expression and found that CD10 demonstrated a pattern of expression in hormone sensitive but not hormone refractory cell lines. When CD10 expression was examined in a tissue microarray, CD10 expression was below the 25th percentile of matched normal prostate tissue in 68% of prostate cancers, below the median expression of matched normal prostate tissue in 86% of cancers, and completely absent in 34% of cancers. Samples of prostatic intraepithelial neoplasia demonstrated CD10 expression that was intermediate between normal prostatic tissue and prostate cancer. Among prostate cancer patients, CD10 expression did not correlate with Gleason score, pathological stage, or biochemical recurrence following radical prostatectomy. CONCLUSIONS: These findings demonstrate that loss or decreased expression of CD10 is an early and frequent event in human prostate cancer and implicates CD10 as a potential therapeutic target for early stage hormone sensitive prostate cancer.

Asunto(s)

Neprilisina/biosíntesis , Neoplasias de la Próstata/enzimología , Anciano , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/patología , Neprilisina/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Análisis por Matrices de Proteínas , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

PURPOSE: We developed a prostate cancer tumor model capable of being noninvasively imaged using positron emission tomography (PET) based on expression of the herpes simplex virus thymidine kinase (HSV1-tk) reporter gene. MATERIALS AND METHODS: The androgen independent, metastatic prostate cancer cell lines CL1 and CL1-GFP were stably transfected with the mutant HSV1-tk gene pcDNA3.1/pCMV-sr39tk, which has increased ability to phosphorylate penciclovir. The presence of the sr39tk gene product was analyzed by Western blot analysis and relative thymidine kinase enzyme activity was assessed by a functional thymidine kinase enzyme activity assay. Subcutaneous and orthotopic CL1 and CL1-SR39 tumor xenografts were established in SCID mice. The ability to image CL1-SR39 was assessed using fluorodeoxyglucose and F-penciclovir ( F-FHBG) micro-PET (a rodent PET scanner). To investigate the systemic distribution of intratumoral sr39tk injections established CL1 tumors were transiently injected with first generation adenoviral vectors carrying the sr39tk gene under control of the strong cytomegalovirus promoter Ad-CMV-HSV1-sr39tk and imaged using micro-PET. RESULTS: Transfection of sr39tk into CL1 cells was successful. CL1-SR39 thymidine kinase enzyme activity was greater than twice the activity of the glioma cell line C6-SR39 control and above the threshold necessary for micro-PET detection. Fluorodeoxyglucose micro-PET in SCID mice was positive for CL1 and CL1-SR39 tumors. Selective micro-PET of subcutaneous CL1-SR39 tumors was done using F-FHBG. Micro-PET imaging after systemic and intratumoral injection of Ad-CMV-HSV1-sr39tk revealed significant systemic transgene leakage with significant hepatic expression of sr39TK protein. CONCLUSIONS: Molecular based imaging of sr39tk transfected prostate cancer tumors and adenoviral delivered HSV1-tk suicide gene therapy based on the selective conversion and intracellular trapping of F-FHBG by sr39tk is feasible. Potential applications include noninvasive monitoring of the location, duration and intensity of gene constructs, which may contribute to the safety of clinical gene therapy protocols, and noninvasive imaging of the prostate cancer xenograft response to experimental therapy.

Asunto(s)

Aciclovir/análogos & derivados , Terapia Genética , Neoplasias de la Próstata/terapia , Tomografía Computarizada de Emisión , Aciclovir/metabolismo , Animales , Western Blotting , Fluorodesoxiglucosa F18 , Genes Reporteros , Vectores Genéticos , Guanina , Herpesvirus Humano 1/genética , Humanos , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/enzimología , Radiofármacos , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Transfección , Células Tumorales Cultivadas