Géza Sáfrány

Detailed characterization of the mouse glioma 261 tumor model for experimental glioblastoma therapy

Mouse glioma 261 (Gl261) cells are used frequently in experimental glioblastoma therapy; however, no detailed description of the Gl261 tumor model is available. Here we present that Gl261 cells carry point mutations in the K‐ras and p53 genes. Basal major histocompatibility complex (MHC)I, but not MHCII, expression was detected in Gl261 cells. The introduction of interferon‐γ‐encoding genes increased expression of both MHCI and MHCII. A low amount of B7‐1 and B7‐2 RNA was detected in wild‐type cells, but cytokine production did not change expression levels. Gl261 cells were transduced efficiently by adenoviral vectors; the infectivity of retroviral vectors was limited. Low numbers of transplanted Gl261 cells formed both subcutaneous and intracranial tumors in C57BL/6 mice. The cells were moderately immunogenic: prevaccination of mice with irradiated tumor cells 7 days before intracranial tumor challenge prevented tumor formation in approximately 90% of mice. When vaccination was carried out on the day or 3 days after tumor challenge, no surviving animals could be found. In vitro‐growing cells were radiosensitive: less than 2 Gy was required to achieve 50% cell mortality. Local tumor irradiation with 4 Gy X‐rays in brain tumor‐bearing mice slowed down tumor progression, but none of the mice were cured off the tumor. In conclusion, the Gl261 brain tumor model might be efficiently used to study the antitumor effects of various therapeutic modalities, but the moderate immunogenicity of the cells should be considered. (Cancer Sci 2006; 97: 546 – 553)

Effects of Low-Dose Radiation on the Immune System of Mice after Total-Body Irradiation

Abstract The effects of acute exposure to low- and high-dose radiation on the quantitative and functional parameters of the immune system were analyzed. C57BL/6 mice were irradiated with different doses of γ radiation (0.01, 0.05, 0.1, 0.5 and 2 Gy) and splenocytes were isolated at various times. Alterations in the distribution and surviving fraction of splenocyte subsets such as CD4+ and CD8+ T lymphocytes, regulatory T cells (Treg), natural killer (NK) cells, dendritic cells (DCs) and B lymphocytes were analyzed by flow cytometry. Apoptosis frequency was quantified by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method 4 h after irradiation. Cytokine expression was investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). Low doses decreased apoptosis in the splenocyte subpopulations studied most prominently in NK cells and DCs. Exposure to 2 Gy increased apoptosis in all splenocyte subpopulations; B cells were the most sensitive and NK cells and DCs the least sensitive. The lowest cell numbers were measured 3 days after irradiation, with minor changes by day 7. CD8+ and B cells were rather resistant to low doses but were very sensitive to 2 Gy, while NK cells, DCs and Treg cells were much more resistant to high doses. Expression of the T-helper 1 (Th1)- and helper 2 (Th2)-type cytokines decreased after low doses and increased after high doses. Interleukin 6 (IL-6) reacted at early times and IL-10 at later times. IL-5 levels were consistently elevated. These data highlight the differences in the responses of different splenocyte subpopulations to low- and high-dose radiation.

Local tumor irradiation augments the antitumor effect of cytokine-producing autologous cancer cell vaccines in a murine glioma model

The combined therapeutic effect of cytokine-producing cancer cell vaccines and local radiotherapy was studied in a mouse glioma 261 (Gl261) brain tumor model. Brain tumor–bearing mice were treated with cytokine (IL-4, IL-6, IL-7, GM-CSF, TNF-α, LIF, LT) producing vaccines made by in vitro transduction of Gl261 cells with the corresponding adenoviral vectors. Vaccines producing either IL-4 or GM-CSF cured 20–40% of mice. The antitumor effect strongly depended on the secreted cytokine level. Vaccination therapy induced specific activation of cytotoxic T lymphocytes measured by cell-mediated cytotoxicity assay. Brain tumors were heavily infiltrated by CD4+ lymphocytes after treatment with IL-4– or GM-CSF–secreting cells. GM-CSF vaccination induced moderate CD8+ infiltration, as well. Depleting either CD4+ or CD8+ lymphocyte subsets abolished the anticancer effect of GM-CSF–expressing cells. Strong synergism was observed by combining cytokine vaccination (GM-CSF, IL-4, IL-12) with local tumor irradiation: about 80–100% of the glioma-bearing mice was cured. The high efficiency of combined treatment was maintained even under suboptimal conditions when neither of the modalities cured any of the mice alone. This suggests that vaccination therapy might open a new potential in the clinical treatment of high-grade gliomas when applied as adjuvant to existing treatment modalities.

The impact of radiation therapy on the antitumor immunity: local effects and systemic consequences.

The main antitumor efficacy of irradiation relies in its direct cytotoxic effect. Increasing evidence indicates a systemic effect of radiation though, mediated mainly by the immune system. In this review we wish to focus on the radiotherapy induced modifications of the soluble and cellular mediators of the antitumor immune response and summarize some of the mechanisms by which radiation driven local and systemic bystander effects can influence tumor immunogenicity. In different tumor types due to the intrinsic immunogenicity of the tumor cells and the immunological characteristics of the tumor microenvironment, different radiation induced immune modulatory mechanisms are predominant. Radiation most probably can only amplify or augment a pro-immunogenic phenotype and can hardly change by itself a net immune suppressing environment into an immune stimulating one. This immune modulatory potential of radiotherapy could be exploited in tumor treatment by developing combined radiotherapeutic and immunotherapeutic approaches. The last few years showed a dramatic increase in the knowledge of radiation induced out-of field and systemic effects, which foresees a rapid progress in the development and clinical application of these new, combined therapies for cancer cure.

A Review on Radiogenic Lhermitte's Sign *

Radiation myelopathy is a rare, but extremely serious side-effect of radiotherapy. Recovery from radiation-induced motor sequelae is rare, whereas, the regeneration of sensory losses is relatively frequent. Among the sensory radiogenic injuries of the spinal cord, Lhermitte’s sign (LS) is most frequent. This review describes the clinical picture and diagnostic imaging signs of radiogenic LS. There have been only a few studies on large patient groups with radiogenic LS, demonstrating a rate of occurrence of 3.6–13%, relating mainly to mantle irradiation or the radiotherapy of head and neck tumors. These cases typically manifest themselves 3 months following radiotherapy and gradually disappear within 6 months. Only 3 LS cases have been described in the English literature with extraordinarily severe symptoms lasting for more than 1 year. MRI, a sensitive tool in the detection of demyelination, failed to reveal any pathological sign accompanying radiogenic LS. However, positron emission tomography demonstrated increased [18F]fluorodeoxyglucose accumulation and [15O]butanol perfusion, but a negligible [11C]methionine uptake in the irradiated spinal cord segments in patients with long-standing LS. These imaging data are suggestive of a close direct relationship between the regional perfusion and metabolism of the spinal cord, very much like the situation in the brain. We postulate that an altered, energy-demanding conduction along the demyelinated axons of patients with chronic radiogenic LS may explain the increased metabolism and perfusion.

Cancer gene therapy: combination with radiation therapy and the role of bystander cell killing in the anti-tumor effect.

Current anti-cancer modalities such as surgery, chemo- and radiation therapies have only limited success in cancer treatment. Gene therapy is a promising new tool to improve outcomes. In this review, first we summarize the various strategies to kill tumor cells, and then focus on the bystander effect of gene therapy. A variety of strategies, such as gene-directed enzyme pro-drug therapy, activation of an anti-tumor immune attack, application of replication-competent and oncolytic viral vectors, tumor-specific as well as radiation and hypoxiainduced gene expression, might be applied to target tumor cells. We put special emphasis on the combination of these approaches with local tumor irradiation. Using the available vector systems, only a small portion of cancer cells contains the therapeutic genes under clinical situations. However, cells directly targeted by gene therapy will transfer death signals to neighboring cancer cells. This bystander cell killing improves the efficiency of cancer gene therapy. Death signals are delivered by cell-to-cell communication through gap junction intercellular contacts, release of toxic metabolites into the neighborhood or to larger distances, phagocytosis of apoptotic bodies, and the activation of the immune system. Bystander cell killing can be enhanced by the introduction of gap junction proteins into cells, by further activating the immune system with immune-stimulatory molecules, or by introducing genes that help the transfer of cytotoxic genes and/or metabolites into bystander cells. In conclusion, although bystander cell killing can improve therapeutic effects, there should be additional developments in cancer gene therapy for a more efficient clinical application.(Pathology Oncology Research Vol 12, No 2, 118–124)

The effect of ionizing radiation on regulatory T cells in health and disease

Treg cells are key elements of the immune system which are responsible for the immune suppressive phenotype of cancer patients. Interaction of Treg cells with conventional anticancer therapies might fundamentally influence cancer therapy response rates. Radiotherapy, apart from its direct tumor cell killing potential, has a contradictory effect on the antitumor immune response: it augments certain immune parameters, while it depresses others. Treg cells are intrinsically radioresistant due to reduced apoptosis and increased proliferation, which leads to their systemic and/or intratumoral enrichment. While physiologically Treg suppression is not enhanced by irradiation, this is not the case in a tumorous environment, where Tregs acquire a highly suppressive phenotype, which is further increased by radiotherapy. This is the reason why the interest for combined radiotherapy and immunotherapy approaches focusing on the abrogation of Treg suppression has increased in cancer therapy in the last few years. Here we summarize the basic mechanisms of Treg radiation response both in healthy and cancerous environments and discuss Treg-targeted pre-clinical and clinical immunotherapy approaches used in combination with radiotherapy. Finally, the discrepant findings regarding the predictive value of Tregs in therapy response are also reviewed.

Phenotypic Profiling of Engineered Mouse Melanomas with Manipulated Histamine Production Identifies Histamine H2 Receptor and Rho-C as Histamine-Regulated Melanoma Progression Markers

In the present study, the impact of acquired neoplastic L-histidine decarboxylase (HDC) expression, and its direct consequence, the release of histamine in the tumor environment, was assessed on melanoma tumor progression. B16-F10 mouse melanoma cells were manipulated via stable transfection, and nine novel transgenic variants were generated in triplicates, constitutively expressing the full-length sense mouse HDC mRNA, a mock control, and an antisense HDC RNA segment, respectively. Establishing both primary skin tumors and lung metastases in C57BL/6 mice, the nine variants with different histamine-releasing capacities were subjected to a comprehensive comparative progression profiling in vivo. Our analyses showed trends of markedly accelerated tumor growth (P < 0.001), and moderately increased metastatic colony-forming potential (P = 0.010) along with rising levels of local histamine production. Using RNase protection assay for screening of the melanoma progression profile, and Western blotting for subsequent result validation, we looked for molecular progression markers affected by melanoma histamine secretion. Investigation of 21 functionally clustered markers associated with tumor proliferation, angiogenesis, invasivity, metastasis formation, local or systemic immunomodulation, and histamine signaling revealed positive correlations between histamine production, tumor histamine H2 receptor and rho-C expression (P < 0.001, P = 0.002, respectively). These observations confirm the involvement of histamine in the molecular machinery of melanoma progression.

Structural analysis of oval-cell–mediated liver regeneration in rats†

We have analyzed the architectural aspects of progenitor‐cell–driven regenerative growth in rat liver by applying the 2‐acetaminofluorene/partial hepatectomy experimental model. The regeneration is initiated by the proliferation of so‐called oval cells. The oval cells at the proximal tips of the ductules have a more differentiated phenotype and higher proliferative rate. This preferential growth results in the formation of a seemingly random collection of small hepatocytes, called foci. These foci have no clonal origin, but possess a highly organized structure, which shows similarities to normal hepatic parenchyma. Therefore, they can easily remodel into the lobular structure. Eventually, the regenerated liver is constructed by enlarged hepatic lobules; no new lobules are formed during this process. The foci of the Solt‐Farber experimental hepatocarcinogenesis model have identical morphological features; accordingly, they also represent only regenerative, not neoplastic, growth. Conclusion: Progenitor‐cell–driven liver regeneration is a well‐designed, highly organized tissue reaction, and better comprehension of the architectural events may help us to recognize this process and understand its role in physiological and pathological reactions. (HEPATOLOGY 2012)

PET identifies transitional metabolic change in the spinal cord following a subthreshold dose of irradiation.

Positron emission tomographic (PET) investigations were performed to obtainin vivo information on symptomless radiation-induced pathological changes in the human spinal cord. PET investigations were carried out prior to radiotherapy and during the regular follow-up in an early hypopharyngeal cancer patient (the spinal cord was irradiated with a biologically effective dose of 80 Gy2), with [18F]fluorodeoxyglucose (FDG), [11C]methionine and [15O]butanol as tracers; radiosensitivity and electroneuronographic (ENG) studies were also performed. A very low background FDG accumulation (mean standardized uptake values, i.e. SUV: 0.84) was observed in the spinal cord before the initiation of radiotherapy. An increased FDG uptake was measured 2 months after the completion of radiotherapy (mean SUV: 1.69), followed by a fall-off, as measured 7 months later (mean SUV: 1.21). By 44 months after completion of irradiation, the FDG accumulation in the irradiated segments of the spinal cord had decreased to a level very close to the initial value (mean SUV: 1.11). The simultaneous [15O]butanol uptake results demonstrated a set of perfusion changes similar to those observed in connection with the FDG accumulation. The patient exhibited an extremely low [11C]methionine uptake within the irradiated and the nonirradiated spinal cord during the clinical course. She has not had any neurological symptoms, and the results of central ENG measurements before radiotherapy and 2 months following its completion proved normal. Radiobiological investigations did not reveal unequivocal signs of an increased radiosensitivity. A transitory increased spinal cord FDG uptake following radiotherapy may be related to the posttherapeutic mild inflammatory and regenerative processes. The normal [11C]methionine accumulation observed is strong evidence against intensive cell proliferation. The high degree of normalization of the temporarily increased FDG uptake of the irradiated spinal cord segments by 44 months is in good agreement with the results of monkey studies, which demonstrated a nearly complete recovery from radiation-induced spinal cord injury.