Interferon- and STING-independent induction of type I interferon stimulated genes during fractionated irradiation.

BACKGROUND: Improvement of radiotherapy efficacy requires better insight in the dynamic responses that occur during irradiation. Here, we aimed to identify the molecular responses that are triggered during clinically applied fractionated irradiation. METHODS: Gene expression analysis was performed by RNAseq or microarray analysis of cancer cells or xenograft tumors, respectively, subjected to 3-5 weeks of 5 × 2 Gy/week. Validation of altered gene expression was performed by qPCR and/or ELISA in multiple cancer cell lines as well as in pre- and on-treatment biopsies from esophageal cancer patients ( NCT02072720 ). Targeted protein inhibition and CRISPR/Cas-induced gene knockout was used to analyze the role of type I interferons and cGAS/STING signaling pathway in the molecular and cellular response to fractionated irradiation. RESULTS: Gene expression analysis identified type I interferon signaling as the most significantly enriched biological process induced during fractionated irradiation. The commonality of this response was confirmed in all irradiated cell lines, the xenograft tumors and in biopsies from esophageal cancer patients. Time-course analyses demonstrated a peak in interferon-stimulated gene (ISG) expression within 2-3 weeks of treatment. The response was accompanied by a variable induction of predominantly interferon-beta and/or -lambda, but blocking these interferons did not affect ISG expression induction. The same was true for targeted inhibition of the upstream regulatory STING protein while knockout of STING expression only delayed the ISG expression induction. CONCLUSIONS: Collectively, the presented data show that clinically applied fractionated low-dose irradiation can induce a delayed type I interferon response that occurs independently of interferon expression or STING signaling. These findings have implications for current efforts that aim to target the type I interferon response for cancer treatment.

Experimental and clinical studies on radiation and curcumin in human glioma.

PURPOSE: There is progressing evidence for the anti-cancer potential of the natural compound and dietary spice curcumin. Curcumin has been ascribed to be cytotoxic for various tumour cell types, to inhibit cell proliferation and to interfere with the cellular oxidant status. The compound has been notified as a therapeutic agent with radiosensitizing potential in brain tumour therapy. We considered the rationale to combine curcumin with radiation in the treatment of human glioblastoma multiforme (GBM). METHOD: Determination of clonogenic cell survival following exposure of U251 human glioma cells to single dose (1-6 Gy) and fractionated irradiation (5 daily fractions of 2 Gy) without and with curcumin. Additional literature search focused on the interaction between curcumin and radiotherapy in experimental and clinical studies on human glioma. RESULTS: No interaction was found on the survival of U251 human glioma cells after irradiation in combination with curcumin at clinically achievable concentrations. Experimental in vitro and in vivo data together with clinical bioavailability data from the literature do not give evidence for a radiosensitizing effect of curcumin. Reported GBM intratumoural curcumin concentrations are too low to either exert an own cytotoxic effect or to synergistically interact with radiation. Novel approaches are being explored to increase the bioavailability of curcumin and to facilitate transport over the blood-brain barrier, aimed to reach therapeutic curcumin levels at the tumour site. CONCLUSION: There is neither a biological nor clinical rationale for using curcumin as radiosensitizer in the therapy of GBM patients.

Development of transient radioresistance during fractionated irradiation in vitro.

BACKGROUND AND PURPOSE: Effective combination treatments with fractionated radiotherapy rely on a proper understanding of the dynamic responses that occur during treatment. We explored the effect of clinical fractionated radiotherapy on the development and timing of radioresistance in tumor cells. METHODS AND MATERIALS: Different colon (HT29/HCT116/COLO320/SW480/RKO) and high-grade astrocytoma (D384/U-251MG) cancer cell lines were treated for 6 weeks with daily fractions of 2 Gy, 5 days per week. Clonogenic survival was determined throughout the treatment period. In addition, the radiosensitivity of irradiated and non-irradiated was compared. Finally, the effect of different dose fractions on the development of radioresistance was determined. RESULTS: All cell lines developed radioresistance within 2-3 weeks during fractionated radiotherapy. This was characterized by the occurrence of a steady state phase of clonogenic survival. In U-251MG cells this was accompanied by increased cell senescence and stemness. After recovering from six weeks of treatment, the radiosensitivity of fractionally irradiated and non-irradiated cells was similar. Including transient radioresistance, described as (α/ß)-(d+1), as a factor in the classic LQ model resulted in a perfect fit with the experimental data observed during fractionated radiotherapy. This was confirmed when different dose fractions were applied. CONCLUSIONS: Fractionated irradiation of cancer cells in vitro following clinical radiation schedules induces a reversible radioresistance response. This adaptive response can be included in the LQ model as a function of the dose fraction and the alpha/beta-ratio of a given cell line. These findings warrant further investigation of the mechanisms and clinical relevance of adaptive radioresistance.

Identification of MEK162 as a Radiosensitizer for the Treatment of Glioblastoma.

Glioblastoma (GBM) is a highly aggressive and lethal brain cancer type. PI3K and MAPK inhibitors have been studied preclinically in GBM as monotherapy, but not in combination with radiotherapy, which is a key component of the current standard treatment of GBM. In our study, GBM cell lines and patient representative primary cultures were grown as multicellular spheroids. Spheroids were treated with a panel of small-molecule drugs including MK2206, RAD001, BEZ235, MLN0128, and MEK162, alone and in combination with irradiation. Following treatment, spheroid growth parameters (growth rate, volume reduction, and time to regrow), cell-cycle distribution and expression of key target proteins were evaluated. In vivo, the effect of irradiation (3 × 2 Gy) without or with MEK162 (50 mg/kg) was studied in orthotopic GBM8 brain tumor xenografts with endpoints tumor growth and animal survival. The MAPK-targeting agent MEK162 was found to enhance the effect of irradiation as demonstrated by growth inhibition of spheroids. MEK162 downregulated and dephosphorylated the cell-cycle checkpoint proteins CDK1/CDK2/WEE1 and DNA damage response proteins p-ATM/p-CHK2. When combined with radiation, this led to a prolonged DNA damage signal. In vivo data on tumor-bearing animals demonstrated a significantly reduced growth rate, increased growth delay, and prolonged survival time. In addition, RNA expression of responsive cell cultures correlated to mesenchymal stratification of patient expression data. In conclusion, the MAPK inhibitor MEK162 was identified as a radiosensitizer in GBM spheroids in vitro and in orthotopic GBM xenografts in vivo The data are supportive for implementation of this targeted agent in an early-phase clinical study in GBM patients. Mol Cancer Ther; 17(2); 347-54. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

The radiosensitizing effect of fluorocyclopentenyl-cytosine (RX-3117) in ovarian and lung cancer cell lines.

RX-3117 (fluorocyclopentenyl-cytosine) is a novel cytidine analog currently being evaluated in a Phase Ib clinical trial in cancer patients with solid tumors. The radiosensitizing effect of RX-3117 was studied in A2780 ovarian cancer cells and non-small cell lung cancer cell lines and related to cell survival and the effect on cell cycle and cell cycle proteins. RX-3117 has a schedule-dependent radiosensitizing effect, but only at pre-incubation (dose modifying factors: 1.4-1.8), observed at pulse and fractionated irradiation. Radiosensitizion was also seen in a three-dimensional spheroid model. At the low radiosensitizing concentration, RX-3117 in combination with radiation led to an accumulation of cells in S-phase, which was accompanied with an increase of cell cycle proteins such as p-Chk2 and p-cdc25C. In addition, RX-3117 caused DNA damage and increased apoptosis. In conclusion, our in vitro experiments showed a radiosensitizing effect of RX-3117.

Low dose angiostatic treatment counteracts radiotherapy-induced tumor perfusion and enhances the anti-tumor effect.

The extent of tumor oxygenation is an important factor contributing to the efficacy of radiation therapy (RTx). Interestingly, several preclinical studies have shown benefit of combining RTx with drugs that inhibit tumor blood vessel growth, i.e. angiostatic therapy. Recent findings show that proper scheduling of both treatment modalities allows dose reduction of angiostatic drugs without affecting therapeutic efficacy. We found that whilst low dose sunitinib (20 mg/kg/day) did not affect the growth of xenograft HT29 colon carcinoma tumors in nude mice, the combination with either single dose RTx (1x 5Gy) or fractionated RTx (5x 2Gy/week, up to 3 weeks) substantially hampered tumor growth compared to either RTx treatment alone. To better understand the interaction between RTx and low dose angiostatic therapy, we explored the effects of RTx on tumor angiogenesis and tissue perfusion. DCE-MRI analyses revealed that fractionated RTx resulted in enhanced perfusion after two weeks of treatment. This mainly occurred in the center of the tumor and was accompanied by increased tissue viability and decreased hypoxia. These effects were accompanied by increased expression of the pro-angiogenic growth factors VEGF and PlGF. DCE-MRI and contrast enhanced ultrasonography showed that the increase in perfusion and tissue viability was counteracted by low-dose sunitinib. Overall, these data give insight in the dynamics of tumor perfusion during conventional 2 Gy fractionated RTx and provide a rationale to combine low dose angiostatic drugs with RTx both in the palliative as well as in the curative setting.

Genome-wide siRNA Screen Identifies the Radiosensitizing Effect of Downregulation of MASTL and FOXM1 in NSCLC.

Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non-small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule-associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggesting that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradiation showed that a number of cell-cycle-related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2-M phase arrest and defects in cytokinesis, which are followed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy.

Radiosensitization by thymidine phosphorylase inhibitor in thymidine phosphorylase negative and overexpressing bladder cancer cell lines.

TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 µM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 µM TPI did not or slightly affect radiosensitivity, but 100 µM TPI alone enhanced the radiation response (p<.05). TFT alone at 1 µM and in combination with 10 µM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ϒH2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.

A new mathematical pharmacodynamic model of clonogenic cancer cell death by doxorubicin.

Previous models for predicting tumor cell growth are mostly based on measurements of total cell numbers. The purpose of this paper is to provide a new simple mathematical model for calculating tumor cell growth focusing on the fraction of cells that is clonogenic. The non-clonogenic cells are considered to be relatively harmless. We performed a number of different types of experiments: a long-term drug "treatment", several concentrations/fixed time experiments and time-series experiments, in which human MCF-7 breast cancer cells were exposed to doxorubicin and the total number of cells were counted. In the latter two types, at every measurement point a plating efficiency experiment was started. The final number of colonies formed is equal to the number of clonogenic cells at the onset of the experiment. Based on the intracellular drug concentration, our model predicts cell culture effects taking clonogenic ability and growth inhibition by neighboring cells into account. The model fitted well to the experimental data. The estimated damage parameter which represents the chance of an MCF-7 cell to become non-clonogenic per unit time and per unit intracellular doxorubicin concentration was found to be 0.0025 ± 0.0008 (mean ± SD) nM(-1) h(-1). The model could be used to calculate the effect of every doxorubicin concentration versus time (C-t) profile. Although in vivo parameters may well be different from those found in vitro, the model can be used to predict trends, e.g. by comparing effects of different in vivo C-t profiles.

Comparable cell survival between high dose rate flattening filter free and conventional dose rate irradiation.

PURPOSE: Investigation of clonogenic cell survival and cell proliferation following single dose and fractionated delivery of high dose rate flattening filter free (FFF) irradiation compared to conventional dose rates. MATERIAL AND METHODS: The human astrocytoma D384, glioma T98 and lung carcinoma SW1573 cell lines were irradiated using either a single dose (0-12 Gy) or a fractionated protocol of 5 daily fractions of 2 Gy (D384) or 3 Gy (SW1573). Cells were irradiated inside a phantom using fixed gantry beams of a linear accelerator. A sliding window technique created homogeneous dose distributions over the surface of the cell cultures. Irradiations using standard beams (6 MV, 600 MU/min.) and high dose rate FFF beams (10 MV, 2400 MU/min.) were compared. Cell survival was determined by clonogenic assay. In the fractionated irradiation set-up, the number of clonogenic cells was estimated by including tumor cell proliferation during the overall treatment time in the analysis. RESULTS: All cell lines showed equal cell survival following irradiation using either the FFF beams or conventional flattened (FF) beams. This was observed after single dose exposure (0-12 Gy) as well as after fractionated irradiation (p = 0.08 for D384 and 0.20 for SW1373 cell lines). CONCLUSION: FFF irradiation with a dose rate of 2400 MU/min and four times higher dose per pulse compared to irradiation with FF beams did not change cell survival for three human cancer cell lines up to a fraction dose of 12 Gy compared to irradiation using FF beams.

Valproic acid sensitizes human glioma cells for temozolomide and γ-radiation.

Temozolomide (TMZ) is given in addition to radiotherapy in glioma patients, but its interaction with the commonly prescribed antiepileptic drug valproic acid (VPA) is largely unknown. Induction of DNA demethylation by VPA could potentially induce expression of the O(6)-methylguanine-DNA-methyltransferase (MGMT) protein, causing resistance to TMZ and thereby antagonizing its effect. Therefore, this study investigates the interaction between VPA, TMZ, and γ-radiation. Two glioma cell lines were used that differ in TMZ sensitivity caused by the absence (D384) or presence (T98) of the MGMT protein. VPA was administered before (24/48 h) or after (24 h) single doses of γ-radiation; or, after 24 h, VPA treatment was accompanied by a single dose of TMZ for another 24 h. For trimodal treatment the combination of VPA and TMZ was followed by single doses of γ-radiation. In both cell lines VPA caused enhancement of the radiation response after preincubation (DMF(0.2) 1.4 and 1.5) but not after postirradiation (DMF(0.2) 1.1 and 1.0). The combination of VPA and TMZ caused enhanced cytotoxicity (DMF(0.2) 1.7) in both the TMZ-sensitive cell line (D384) and the TMZ-resistant cell line (T98). The combination of VPA and TMZ caused a significant radiation enhancement (DMF(0.2) 1.9 and 1.6) that was slightly more effective than that of VPA alone. VPA does not antagonize the cytotoxic effects of TMZ. Preincubation with VPA enhances the effect of both γ-radiation and TMZ, in both a TMZ-sensitive and a TMZ-resistant human glioma cell line. VPA combined with TMZ may lead to further enhancement of the radiation response.

Anti-tumour effects by a trimodal combination of temozolomide, meloxicam and X-rays in cultures of human glioma cells.

PURPOSE: To investigate the possible cytotoxic interactions between the chemotherapeutic drug temozolomide (TMZ) and the cyclooxygenase-2 inhibitor meloxicam (MLC) or of both drugs combined with X-rays in three human glioma cell lines (D384, Hs 683 and U251). MATERIALS AND METHODS: Cells were exposed to TMZ (96 hours) and MLC was co-incubated during the last 24 h. Thereafter, cells were irradiated with X-rays and plated for a clonogenic assay. Total cell numbers and the numbers of surviving cells were determined to study the recovery of the cell populations (up until 19 days) following different combinations of TMZ, MLC and X-rays. RESULTS: The combination of MLC and TMZ caused an enhanced cytotoxic effect in D384 and Hs 683. Various treatment combinations demonstrated significant radiation enhancement in all three cell lines. Long-term observations of D384 cells demonstrated that the repopulation rates of the surviving cells are far less affected by the various treatment protocols than those from the non-surviving cells. CONCLUSIONS: The present study demonstrates that a combination of TMZ and MLC resulted in a significant potentiation of their cytotoxicity in D384 and Hs683. The combination of these two drugs can also cause considerable enhancement of the radiation response in human glioma cell lines, although only D384 cells benefit from trimodal over bimodal treatment.

Strategies for the analysis of in vitro radiation sensitivity and prediction of interaction with potential radiation modifying agents.

PURPOSE: To better predict radiation-drug interactions in in vitro model systems, thorough assessment of the effects of in vitro exposure is required. The aim of this article is to show that both clonogenic capacity and cellular proliferation, which represent important different elements of tumour conduct, can be considered when assessing in vitro radio sensitisation. METHODS: A model was designed that can predict radiation-drug interactions based on changes in clonogenic capacity and cell proliferation by radiation modifying agents. RESULTS: Using this mechanistical model, the effect of combined exposure to radiation and potential drugs can be tested on both established cell lines and primary cells. In addition, we could obtain more information about the mechanisms underlying the radiation-drug interaction by assessing the results of in vitro exposure on tumour cell proliferation and clonogenic capacity according to our model. CONCLUSIONS: The significance of our model is not to replace the clonogenic gold standard but to give additional information about the radiation-drug combination by determining cell proliferation. Moreover, the advantage is that the interaction can also be predicted in cases where a clonogenic assay is not possible. Additional research into the biological effect of potential radio-sensitisers is warranted for future (pre)clinical studies.

Depth profile characterization of ultra shallow junction implants.

A need for analysis techniques, complementary to secondary ion mass spectrometry (SIMS), for depth profiling dopants in silicon for ultra shallow junction (USJ) applications in CMOS technologies has recently emerged following the difficulties SIMS is facing there. Grazing incidence X-ray fluorescence (GIXRF) analysis in the soft X-ray range is a high-potential tool for this purpose. It provides excellent conditions for the excitation of the B-K and the As-L(iii,ii) shells. The X-ray standing wave (XSW) field associated with GIXRF on flat samples is used here as a tunable sensor to obtain information about the implantation profile because the in-depth changes of the XSW intensity are dependent on the angle of incidence. This technique is very sensitive to near-surface layers and is therefore well suited for the analysis of USJ distributions. Si wafers implanted with either arsenic or boron at different fluences and implantation energies were used to compare SIMS with synchrotron radiation-induced GIXRF analysis. GIXRF measurements were carried out at the laboratory of the Physikalisch-Technische Bundesanstalt (PTB) at the electron storage ring BESSY II using monochromatized undulator radiation of well-known radiant power and spectral purity. The use of an absolutely calibrated energy-dispersive detector for the acquisition of the B-Kalpha and As-Lalpha fluorescence radiation enabled the absolute determination of the total retained dose. The concentration profile was obtained by ab initio calculation and comparison with the angular measurements of the X-ray fluorescence.

Differential radiosensitizing potential of temozolomide in MGMT promoter methylated glioblastoma multiforme cell lines.

PURPOSE: To investigate the radiosensitizing potential of temozolomide (TMZ) for human glioblastoma multiforme (GBM) cell lines using single-dose and fractionated gamma-irradiation. METHODS AND MATERIALS: Three genetically characterized human GBM cell lines (AMC-3046, VU-109, and VU-122) were exposed to various single (0-6 Gy) and daily fractionated doses (2 Gy per fraction) of gamma-irradiation. Repeated TMZ doses were given before and concurrent with irradiation treatment. Immediately plated clonogenic cell-survival curves were determined for both the single-dose and the fractionated irradiation experiments. To establish the net effect of clonogenic cell survival and cell proliferation, growth curves were determined, expressed as the number of surviving cells. RESULTS: All three cell lines showed MGMT promoter methylation, lacked MGMT protein expression, and were sensitive to TMZ. The isotoxic TMZ concentrations used were in a clinically feasible range of 10 micromol/L (AMC-3046), 3 micromol/L (VU-109), and 2.5 micromol/L (VU-122). Temozolomide was able to radiosensitize two cell lines (AMC 3046 and VU-122) using single-dose irradiation. A reduction in the number of surviving cells after treatment with the combination of TMZ and fractionated irradiation was seen in all three cell lines, but only AMC 3046 showed a radiosensitizing effect. CONCLUSIONS: This study on TMZ-sensitive GBM cell lines shows that TMZ can act as a radiosensitizer and is at least additive to gamma-irradiation. Enhancement of the radiation response by TMZ seems to be independent of the epigenetically silenced MGMT gene.

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells.

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0-6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24-72 h exposure to 250-750 microM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 microM meloxicam for 24 h increased the fraction of cells in the radiosensitive G(2)/M cell cycle phase in D384 (18-27%) and U251 (17-41%) cells. 750 microM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.

Ultralow energy ion beam surface modification of low density polyethylene.

Ultralow energy Ar+ and O+ ion beam irradiation of low density polyethylene has been carried out under controlled dose and monoenergetic conditions. XPS of Ar+-treated surfaces exposed to ambient atmosphere show that the bombardment of 50 eV Ar+ ions at a total dose of 10(16) cm(-2) gives rise to very reactive surfaces with oxygen incorporation at about 50% of the species present in the upper surface layer. Using pure O+ beam irradiation, comparatively low O incorporation is achieved without exposure to atmosphere (approximately 13% O in the upper surface). However, if the surface is activated by Ar+ pretreatment, then large oxygen contents can be achieved under subsequent O+ irradiation (up to 48% O). The results show that for very low energy (20 eV) oxygen ions there is a dose threshold of about 5 x 10(15) cm(-2) before surface oxygen incorporation is observed. It appears that, for both Ar+ and O+ ions in this regime, the degree of surface modification is only very weakly dependent on the ion energy. The results suggest that in the nonequilibrium plasma treatment of polymers, where the ion flux is typically 10(18) m(-2) s(-1), low energy ions (<50 eV) may be responsible for surface chemical modification.

Potential of the conditionally replicative adenovirus Ad5-Delta24RGD in the treatment of malignant gliomas and its enhanced effect with radiotherapy.

The use of replication-competent adenoviruses (Ads) for cancer therapy is receiving widespread attention, especially for the treatment of tumors refractory to current treatments such as glioblastoma. AdDelta24, which carries a 24-bp deletion in E1A and replicates in cells with a retinoblastoma-defective pathway, produced a strong antitumor effect in glioma. To improve infection efficiency of primary glioma cells, which express low levels of coxsackie adenovirus receptor (CAR), the tropism of AdDelta24 was expanded toward alphav integrins by insertion of an Arg-Gly-Asp (RGD) motif into the fiber knob (Ad5-Delta24RGD). We show that Ad5-Delta24RGD had a stronger oncolytic effect than the non-RGD-expressing variant on a broad panel of primary glioma cells, in particular on those with low CAR expression. The effects of Ad5-Delta24RGD were also assessed on a panel of primary organotypic glioma spheroids. In all cases, Ad5-Delta24RGD strongly decreased the viability of these small tumor nodules in vitro. In s.c. glioblastoma xenografts expressing low levels of CAR, five intratumoral injections of 1 x 10(7) plaque-forming units Ad5-Delta24RGD resulted in complete tumor regression in 9 of 10 mice and long-term survival in all treated mice. Preclinical evaluations and clinical trials of replication-competent Ad have shown more promising results when combined with conventional therapeutics. Therefore, we assessed the effects of Ad5-Delta24RGD in combination with radiotherapy. Low-dose irradiation before Ad5-Delta24RGD infection decreased viability of glioma cells more effectively than Ad5-Delta24RGD alone with effects ranging from additive to supra-additive. In addition, combination treatment with Ad5-Delta24RGD and irradiation was studied in glioma xenografts. Five injections of 1 x 10(6) plaque-forming units Ad5-Delta24RGD induced significant tumor growth delay of >119 days compared with untreated controls and led to long-term survival in 6 of 9 mice. When viral treatment was combined with irradiation, tumor regression occurred in all mice resulting in long-term survival without evidence of tumor regrowth in 10 of 10 cases. This study thus provides evidence that Ad5-Delta24RGD has strong antitumor activity in malignant glioma, which can be additionally enhanced by irradiation such that the same therapeutic effect is achieved when a 10-fold lower viral dose is applied. These results support further development of Ad5-Delta24RGD in combination with radiation therapy for treatment of these highly malignant tumors.