Welfare Assessment following Heterotopic or Orthotopic Inoculation of Bladder Cancer in C57BL/6 Mice.

Few studies have assessed whether mice used as cancer models experience pain. Despite this possibility, the usual practice is to withhold analgesics as these are generally viewed as confounding. However, pain also alters cancer progression, so preventing it might not only be beneficial to welfare but also to study validity. Establishing the extent to which different cancer models result in pain is an important first step towards their refinement. We used conditioned place preference (CPP) testing and body-weight and behaviour analyses to evaluate the assumption that heterotopically implanted tumours result in less pain and fewer welfare concerns than those implanted orthotopically. C57Bl/6 mice received MB49Luc luciferase expressing bladder cancer cells or saline implanted subcutaneously or into the bladder. These tumour-bearing or control groups underwent 2 daily 45 minute conditioning trials to saline or morphine (2mg/kg) and then a 15 minute drug-free preference test on day 3 of a 3 day cycle, continuing until the study ended. Tumours were imaged and behaviour data obtained following preference tests. Development of preference for the morphine-paired chamber (morphine-seeking) was determined over time. Heterotopic tumour development had no effect on morphine-seeking, and although the restraint used for heterotopic inoculation caused greater initial weight losses than anaesthesia, these mice steadily gained weight and behaved comparatively normally throughout the study. Orthotopic tumour inoculation caused no initial weight losses, but over the final 7 days these mice became less active and lost more body weight than cancer-free controls. This indicated orthotopic implantation probably caused a more negative impact on welfare or conceivably pain; but only according to the current test methods. Pain could not be confirmed because morphine-seeking in the tumour-bearing groups was similar to that seen in controls. Imaging was not found to be an effective method of monitoring tumour development surpassing manual tumour inspection.

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

Osteopontin and splice variant expression level in human malignant glioma: radiobiologic effects and prognosis after radiotherapy.

BACKGROUND AND PURPOSE: We investigated the role of the hypoxia-associated secreted glycoprotein osteopontin (OPN) in the response of malignant glioma to radiotherapy by characterizing OPN and its splice variants in vitro and in patient material. MATERIAL AND METHODS: The effect of siRNA knockdown of OPN splice variants on cellular and radiobiologic behavior was analyzed in U251MG cells using OpnS siRNA (inhibition of all OPN splice variants) and OpnAC siRNA (knockdown only of OPNa and OPNc). OPN and splice variant mRNA levels were quantified in archival material of 41 glioblastoma tumor samples. Plasma OPN was prospectively measured in 33 malignant glioma patients. RESULTS: Inhibition of OPNa and OPNc (OpnAC) reduced clonogenic survival in U251MG cells but did not affect proliferation, migration or apoptosis. Knockdown of all OPN splice variants (OpnS) resulted in an even stronger inhibition of clonogenic survival, while cell proliferation and migration were reduced and rate of apoptosis was increased. Additional irradiation had additive effects with both siRNAs. Plasma OPN increased continuously in malignant glioma patients and was associated with poor survival. CONCLUSIONS: OPNb is partially able to compensate the effects of OPNa and OPNc knockdown in U251MG cells. High OPN plasma levels at the end of radiotherapy are associated with poor survival.

A redshifted codon-optimized firefly luciferase is a sensitive reporter for bioluminescence imaging.

Bioluminescence imaging has evolved as a powerful tool for monitoring biological processes in vivo. As transmission efficiency of light through tissue increases greatly for wavelengths above 600 nm we examined whether a redshifted codon-optimized firefly luciferase (lambdamax=615 nm) could be successfully employed as a sensitive reporter in mammalian cells. To this end, unmodified codon-optimized luciferase (lambdamax=557 nm) as well as the red-emitting S284T mutant luciferase were expressed simultaneously in human glioma cells in vitro as well as in quadriceps muscles of mice in vivo. We show here that activity of the redshifted enzyme in human glioma cell culture approached approximately one-fourth of that seen with the unmodified enzyme. In contrast, light emission by the red-emitting luciferase in vivo was generally more efficient than that produced by its unmodified counterpart, most likely due to reduced absorption of red light by tissue. The mean ratio of light emission produced by the redshifted luciferase to that of the unmodified enzyme in vivo was approximately 3. Application of this new redshifted luciferase together with other optical reporters may be of considerable importance to biological research as it allows for imaging of deeper tissues as well as simultaneous monitoring of two molecular events in vitro and in vivo if appropriate filter sets are employed.

Tumor-specific activity of cellular regulatory elements is down-regulated upon insertion into the herpes simplex virus genome.

Transcriptional targeting of viral genes is a promising strategy to achieve tumor-specific replication of oncolytic viruses. Due to its natural tropism, herpes simplex virus type 1 (HSV-1) may be an ideal tool for oncolytic therapy of brain tumors such as malignant glioblastoma. To study whether glioma-specific gene expression can be accomplished within the HSV-1 genome, four cellular regulatory elements were exemplarily studied. Whereas the human telomerase reverse transcriptase (hTERT) and survivin promoters and the nestin and vascular endothelial growth factor A (VEGF-A) enhancers displayed pronounced glioma specificity after plasmid transfection, only the nestin enhancer conferred a certain selectivity for glioma cells and notable activity when transferred into the viral genome. The nestin enhancer was also found to be highly useful for tumor cell-specific expression of a therapeutically relevant gene (interleukin-2) when tested in combination with the hTERT or simian virus 40 (SV40) early promoter in the HSV-1 genome. Because activity of the chosen promoter in a tumor is a prerequisite for the successful application of an oncolytic virus, we examined whether the activity of a promoter can be deduced from the amounts of cellular mRNA or protein expressed under its control. We found little correlation between promoter activity and mRNA levels of the corresponding gene, whereas protein expression was more closely related to promoter activity. We conclude that the cellular elements are differently regulated in the viral and cellular genomes. Mechanistic insight into the differential regulation is required to improve and refine the design of transcriptionally targeted HSV vectors.

Bioluminescence imaging to monitor bladder cancer cell adhesion in vivo: a new approach to optimize a syngeneic, orthotopic, murine bladder cancer model.

OBJECTIVE: To improve the orthotopic murine bladder cancer model by using bioluminescent (BL) MB49 tumour cells for noninvasive in vivo monitoring of tumour growth and to examine the efficacy of integrin receptor-blocking oligopeptides on preventing tumour cell adhesion in this improved bladder cancer model. MATERIALS AND METHODS: The capacity of oligopeptide combinations to interfere with tumour cell adhesion was assessed in vivo in a syngeneic, orthotopic, murine bladder cancer model. Tumour outgrowth was monitored noninvasively by bioluminescence imaging (BLI) after administration of luciferase-expressing MB49(LUC) bladder cancer cells. The presence of tumour cells was verified histologically and immunohistochemically on paraffin wax-embedded sections of excised bladders. RESULTS: Anti-adhesive oligopeptides effectively inhibited tumour outgrowth. BLI detected tumour cells at an early stage when there were no clinical signs of cancer in any of the mice. The technique has high sensitivity in detecting tumour cell implantation, but is less reliable in assessing tumour volume in advanced-stage disease due to light attenuation in large tumours. CONCLUSIONS: Peptides targeting adhesion molecules prevent attachment of bladder cancer cells to the injured bladder wall. BLI is a sensitive method for detecting luminescent bladder cancer cells in an orthotopic mouse model.

Autoantibodies to the inhibitor of apoptosis protein survivin in patients with brain tumors.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and is frequently expressed in cancers, including meningiomas and gliomas. Survivin may be associated with tumor progression and poor prognosis of patients with brain tumors. Using ELISA and immunoblot analysis we asked whether survivin is capable of eliciting a humoral immune response in patients with meningiomas and gliomas. Survivin-specific antibodies were detected in 5 of 42 (11.9%) patients with meningiomas and 3 of 35 (8.6%) patients with malignant gliomas of the WHO grades 3 and 4, but not in healthy controls. Tumors of patients with detectable anti-survivin antibodies demonstrated survivin expression in at least 20% of the tumor cells as assessed by immunohistochemistry. We conclude that patients with meningiomas and malignant gliomas can mount a high-titer IgG immune response against the 'universal' tumor-associated antigen survivin. Anti-survivin antibodies may represent attractive tools for diagnosis and follow-up of brain tumors.

Clinical studies with targeted toxins in malignant glioma.

Targeted toxins represent a new class of agents with high specificity for tumor cells. Toxins in current clinical use for the treatment of brain tumors are mostly recombinant polypeptides consisting of a tumor-selective ligand coupled to a peptide toxin of bacterial origin. Targeted toxins are highly potent - one single molecule of toxin is enough to cause cell death. Toxins are able to kill tumor cells independent of any malignancy-associated genetic alterations and/or mutations. The blood-brain barrier has been a major obstacle for using targeted toxins for treatment of malignant glioma. Convection-enhanced delivery (CED), a method for delivery of large molecules to brain tissue via continuous interstitial microinfusion, has permitted direct administration of toxins to brain tumors or to surrounding brain tissue infiltrated by tumor cells. Four targeted toxins advanced to at least phase II clinical trials and are being used for treatment of adult or pediatric patients with recurrent or progressive malignant glioma. These are IL4-P. aeruginosa exotoxin (IL4-PE, NBI-3001), tumor growth factor (TGF)alpha-P. aeruginosa exotoxin (TP-38), IL13-P. aeruginosa exotoxin (IL13-PE38), and transferrin-C. diphtheriae toxin (TransMID(trade mark), Tf-CRM107). All of these toxins have shown an acceptable profile of toxicity and safety in phase I and II clinical studies and have demonstrated some evidence for tumor response. Current phase I and II clinical protocols are exploring several parameters, such as placement of catheters for CED either intratumorally or in the brain tissue surrounding a tumor, surgical resection of tumor before or after toxin infusion, and single vs. repeated infusion. Two large randomized and controlled phase III multicenter studies using IL13-PE38 or TransMID(trade mark) are currently enrolling patients. This review summarizes the study protocols and key findings of all previously completed and currently ongoing clinical studies with targeted toxins for malignant glioma. It offers in addition an outlook into future areas of development of targeted toxins, such as improved delivery modes and non-invasive in vivo imaging of intracerebral and intratumoral distribution of toxin in patients.

Technology evaluation: TransMID, KS Biomedix/Nycomed/Sosei/PharmaEngine.

KS Biomedix (formerly Avicenna Medica; now a subsidiary of the Xenova group) and Nycomed, together with Japanese licensee Sosei and Chinese licensee PharmaEngine, are developing TransMID, a transferrin-mediated diphtheria toxin delivery system for the potential treatment of adult, recurrent, inoperable, high-grade glioma (as TransMID-107R). It is also under investigation for other forms of brain cancer, including early brain cancer (as TransMID-107N), metastatic brain cancer (as TransMID-107M) and pediatric brain cancer (as TransMID-107P). TransMID is currently undergoing phase III clinical trials.

Technology evaluation: cintredekin besudotox, NeoPharm/Nippon.

NeoPharm Inc, under license from the National Institutes of Health and the FDA, and in collaboration with the Japanese licensee Nippon Kayaku Co Ltd, is developing cintredekin besudotox, a chimeric human IL-13 conjugated to a genetically engineered Pseudomonas exotoxin molecule, as a potential antitumor agent. This agent is currently undergoing phase III clinical trials.

Minichromosome maintenance protein 3 elicits a cancer-restricted immune response in patients with brain malignancies and is a strong independent predictor of survival in patients with anaplastic astrocytoma.

PURPOSE: The identification of new molecular markers in astrocytic tumors may help to understand the biology of these tumors in more detail. Informative tumor markers may represent prognostic factors for response to therapy and outcome as well as potential targets for novel anticancer therapies. EXPERIMENTAL DESIGN: Tumor-associated antigens were identified by immunoscreening of a human glioma cDNA expression library with allogeneic sera from patients with diffuse astrocytoma (WHO grades 2-4). The expression of one of the identified antigens, the replication licensing factor minichromosome maintenance protein 3 (MCM3), was analyzed by immunohistochemistry in 142 primary and 27 recurrent astrocytomas (WHO grades 2-4). In addition, 98 serum specimens from patients with primary and secondary brain malignancies and 30 serum specimens from healthy controls were examined by serologic immunoscreening for immunoreactivity with MCM3. RESULTS: MCM3 is overexpressed in human astrocytic tumors and elicits a cancer-restricted humoral immune response in 9.3% (9 of 97) of patients with brain tumors (n = 95) and brain metastases (n = 2) but not in healthy controls. Expression of MCM3 in diffuse astrocytoma is significantly associated with age (P < 0.001), histologic grade (P < 0.001), time to recurrence (P = 0.01), and expression of the proliferation marker Ki-67 (P < 0.001) but not with sex (P = 0.800). Univariate and multivariate Cox regression analysis confirmed MCM3 expression as an independent predictor of poor outcome in astrocytoma patients (P < 0.001 for both). CONCLUSIONS: MCM3 may represent a glioma-associated antigen with significant prognostic role as well as have some potential as a target for cancer-directed therapy.

A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma.

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.

Bioluminescence imaging in vivo - application to cancer research.

Molecular events promoting tumourigenesis and anticancer therapeutic strategies have been intensively studied in tumour cell culture models. In the past few years, non-invasive bioluminescence imaging (BLI) has emerged as a powerful strategy for the validation of cell culture findings in animal models of cancer. BLI allows for repetitive and exceptionally sensitive real-time monitoring of a disease course, as well as of tumour response to therapeutic interventions in an individual animal. This review discusses the application of BLI to cancer research in general and to the area of experimental neuro-oncology in particular.

Intracellular localization of Herpes simplex virus type 1 thymidine kinase fused to different fluorescent proteins depends on choice of fluorescent tag.

Gene therapy employing the suicide gene/prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) is effective in killing malignant tumor cells. Labeling of the HSV-TK enzyme with fluorescent proteins makes possible the non-invasive imaging of transduction efficiency, enzyme localization and activity in cell culture and in animal models of human cancers. Here we report the expression of HSV-TK tagged with different fluorescent proteins (EGFP, DSRed1, DsRed2, dsdrFP616) and show that intracellular localization of the fusion products depends on the nature of the fluorescent tag despite the presence of several nuclear targeting signals within the enzyme itself. Coexpression of red fluorescent HSV-TK fusion proteins with TK-EGFP or untagged HSV-TK allowed these proteins to enter the nucleus by inhibiting formation of red fluorescent protein oligomers. As enzyme localization may influence HSV-TK activity, this observation is of potential importance to gene therapy studies.

An unexpectedly high frequency of hypergalactosemia in an immigrant Bosnian population revealed by newborn screening.

In galactokinase (GALK) deficiency, galactose cannot be phosphorylated into galactose-1-phosphate, which leads to cataract formation. Neonatal screening for hypergalactosemia in Berlin has been performed by thin-layer chromatography since 1978, which detects classical galactosemia and GALK deficiency. Until 1991, GALK deficiency has not been identified in a total of approximately 260,000 samples. In contrast, from 1992 to 1999, nine patients were detected in a total of approximately 240,000 screened newborns. One Turkish patient was homozygous for two novel S142I/G148C GALK mutations in close proximity to the putative ATP-binding site of the enzyme. The other eight children were born to five families belonging to the Bosnian refugee population consisting of approximately 30,000 individuals who have arrived in Berlin since 1991. In two of these families, GALK deficiency was subsequently diagnosed in siblings who had cataract surgery at 4 and 5 y of age, respectively. In all these 10 Bosnian patients, a homozygous P28T mutation located near the active center of the enzyme was identified. We propose that neonatal screening of populations with a significant proportion of Bosnians and possibly other southeastern Europeans, e.g. Romani, should be particularly directed toward GALK deficiency, an inborn error of metabolism that is readily amenable to effective treatment.