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Supplementary Figure 4. Growth of shMASTL cells and the representation of BUB1 knockdown.

Supplementary Table 5. Top 10 enriched clusters from phosphoproteomics analyses.

Supplementary Figure 1. PRKDC knockdown induces radiosensitization.

Supplementary Figure Legends. Legends to the Supplementary Figures

Supplementary Figure 3. MASTL knockdown in A549 cells.

Supplementary Table 2. Proteins phosporylated to a lower extent in SW1573 MASTL knockdown cells in comparison to SW1573 control cells.

Supplementary Table 4. DNA damage cluster obtained from DAVID analysis on proteins upregulated upon exposure to radiation.

Supplemental Figure 1. Chemical structures of non-FDA approved compounds used in the Study; Supplemental Figure 2. Effect of drugs on growth and target phosphorylation; Supplemental Figure 3. mTOR inhibition does not show dose dependent Radiosensitization; Supplemental Figure 4. MEK162 + RT abrogate spheroid regrowth; Supplemental Figure 5. MEK162 radiosensitizes GBM8 primary spheroid culture and increases γH2AX levels; Supplemental Figure 6. MEK162 abrogates BrdU washout and increases sub-G1 accumulation of BrdU+ cells; Supplemental Figure 7. MEK162 + RT in vivo; Supplemental Table 1: Antibodies used; Supplemental Table 2: Gene signature MEK+RT response

Supplementary Table 6. Cell cycle cluster obtained from DAVID analysis on proteins that are less phosphorylated in SW1573 shMASTL cells upon radiation.

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 AACR.See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”

Supplementary Figure 2. Knockdowns of FOXM1 and MASTL achieved by siRNAs or shRNA constructs.

Supplementary Methods. Supplementary methods describing the materials and methods for the phosphoproteomics.

Supplementary Table 3. Proteins phosporylated to a lower extent after 2Gy of ionizing radiation in SW1573 MASTL knockdown cells in comparison to SW1573 control cells.

Supplementary Table 1. Hit list genome wide siRNA screen

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. Mol Cancer Ther; 14(6); 1434–44. ©2015 AACR.

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.

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/β)−( 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.

The immediate surface and sub-surface composition of heat treated La0.6Sr0.4Co0.2Fe0.8O3−δ samples was measured by ion beam analysis and compared to oxygen transport properties over the same depth scale. Consistent with the literature, strontium segregation was observed for samples that received thermal treatments with the formation of an Sr–O based monolayer at the surface. Just below this, a sub-surface strontium depletion region over a depth scale of approximately 2–15 nm was seen. In this sub-surface region, a depletion of lanthanum was also observed. Oxygen transport properties were measured using isotopic labelling techniques and showed that, despite large changes in composition, the transport properties remain largely unchanged. Because strontium diffusion is slow in the bulk and grain boundaries only account for 0.03% of the material (due to large grains), it is suggested that 2D defects (such as dislocations and twins) can act as fast diffusion pathways for the strontium to account for the Sr depletion region observed below the surface.

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.

The process characteristics, the surface chemistry, and the resulting film properties of Ru deposited by atomic layer deposition from (ethylbenzyl)(1-ethyl-1,4-cyclohexadienyl)Ru(0) (EBECHRu) and O2 are discussed. The surface chemistry was characterized by both combustion reactions as well as EBECHRu surface reactions by ligand release. The process behavior on TiN starting surfaces at 325 °C was strongly influenced by Ti(O,N)x segregation on the growing Ru surface with consequences for both the growth per cycle as well as the film properties. For optimized process conditions, the films showed high purity with low C and O concentrations of the order of 1020 at./cm3. Higher deposition temperature led to strong (001) fiber texture of the films on SiO2 starting surfaces. Annealing in forming gas improved the crystallinity and led to resistivity values as low as 11 μΩcm for Ru films with a thickness of 10 nm.

Since the ’70, physics of interaction between bent crystals and heavy particles (protons or ions) [...]

In this study, plasma-enhanced pulsed laser deposition (PE-PLD), which is a novel variant of pulsed laser deposition that combines laser ablation of metal targets with an electrically-produced oxygen plasma background, has been used for the fabrication of ZnO and Cu2O thin films. Samples prepared using the PE-PLD process, with the aim of generating desirable properties for a range of electrical and optical applications, have been analysed using medium energy ion scattering. Using a 100 keV He+ ion beam, high resolution depth profiling of the films was performed with an analysis of the stoichiometry and interface abruptness of these novel materials. It was found that the PE-PLD process can create stoichiometric thin films, the uniformity of which can be controlled by varying the power of the inductively coupled plasma. This technique showed a high deposition rate of ∼ 0.1 nm s−1.

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.

Introduction To improve radiotherapy efficacy, insight in the dynamic biological responses that occur during clinical radiation schedules is vital. In the current study we explored gene expression changes during the course of fractionated radiotherapy (FrRT) that might be targeted with combination treatment strategies. Methods Human cell lines HT29, SW480, HCT116, COLO320, RKO (colorectal carcinoma), D384 (glioblastoma) and OE19 (esophageal adenocarcinoma) as well as HT29 xenograft tumors in nude mice were irradiated up to 3 to 5 weeks with daily fractions of 2 Gy for 5 days per week. In vivo tumor growth was monitored daily and in vitro clonogenic survival was determined every other day. At the end of each treatment week tumors and cells were harvested for RNA isolation. Subsequently, gene expression analysis was performed by RNA sequencing and micro-array analysis. Altered gene expression levels were validated by qPCR. Protein expression was determined by ELISA and Western blot. Results Tumor growth of the HT29 xenografts declined after 2 weeks of FrRT compared to non-irradiated tumors. In irradiated cell lines, clonogenic survival analyses revealed a log-linear decline in survival in the first 2 weeks, after which a steady-state-like phase was reached up to 5 treatment weeks. Gene expression analysis of both xenograft tumors as well as irradiated cell lines at this 2 week time point identified a type-1 interferon (IFN) mediated signaling pathway as the most significantly enriched biological process. The induction of a selection of IFN-stimulated genes (ISGs) was confirmed by qPCR. Comparable responses were observed in both STING positive (HT29, SW480, HCT116, D384) and negative (COLO320, RKO, OE19) cell lines. To get more insight in the dynamics of the IFN response we analyzed ISGs expression at the end of each treatment week. This revealed a peak in ISGs expression after 10 to 15 fractions, both in vivo and in vitro. The ISGs expression levels decreased with continuation of treatment but always remained above the basal level of non-irradiated cells. Single dose irradiation up to 10 Gy also induced dose-dependent ISGs expression, albeit typically 5 to 10 fold lower as compared to FrRT. Subsequent protein and mRNA expression analyses consistently revealed induction of IFN beta expression, with peak levels measured after 10 to 15 fractions. In addition, occasional induction of IFN lambda 2/3 protein and mRNA expression was observed. Conclusion FrRT induces an intrinsic type-1 IFN response which occurs independent of STING. This response is associated with induction of IFN beta expression, which peaks after 2 to 3 weeks of treatment and coincides with a shift in in vivo tumor growth and in vitro clonogenic survival. Targeting this response during FrRT might be exploited as a novel combination treatment. Citation Format: Ruben S. Goedegebuure, Esther A. Kleibeuker, Kitty C. Castricum, Jaap van den Berg, Sarah Derks, Henk M. Verheul, Ben J. Slotman, Adrian Harris, Victor L. Thijssen. A STING independent type-1 interferon response induced by fractionated radiotherapy coincides with altered tumor growth and clonogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3738.

Plasma doping (PLAD) is increasingly applied in microelectronic device manufacture to produce high throughput, high fluence implants. In this medium energy ion scattering (MEIS) study of the PLAD process, Si(100) wafers were exposed to an As containing plasma while pulse biased negatively to 7 kV to cause (recoil) implantation and deposition of As. Quantitative MEIS depth profiling analysis in conjunction with energy spectrum simulation was applied to characterize the near-surface layer changes of the Si wafer following (i) the PLAD process, (ii) two types of chemical wet clean (oxidizing and nonoxidizing), and (iii) spike annealing in an N2 atmosphere. MEIS analysis showed that the PLAD process produced an intermixed As/Si layer, with a near-surface As content of ∼40% that decayed almost linearly to near-zero at a depth of ∼17 nm. This mixed As/Si layer was unstable in air and the initially recorded 1.2 nm thick oxide cap layer grew over a period of four months to 3.5 nm with a concurrent 25% As loss by sublimation. The application of the industry standard, oxidizing wet chemical clean removed the top As and concurrently produced a ∼14 nm thick Si oxide above the remaining implanted As profile, which matched the tail of original As implant profile. As depth profiles measured for the 7 kV PLAD process after a wet clean and spike annealing showed solid phase epitaxial regrowth of the disordered layer. A detailed comparison of the random and aligned MEIS spectra yielded depth profiles of substitutional As with a concentration in excess of 1 × 1021 As cm−3 over a depth greater than 10 nm. The retained dose of 1.35 × 1015 cm−2 represents a ∼70% increase in substitutional As compared to that recorded after a nonoxidizing clean. Such an alternative wet chemical clean, in which Si reoxidation did not occur, was applied to determine the depth of the mixed As/Si layer removed. Found to be 7 nm, the analysis indicated that the etching process ceased when the Si concentration reached 4 × 1022 cm−3. After spike annealing, part of the remaining As had segregated in a thin layer under a 1.6 nm thick surface oxide. The retained As dose in this case was 8 × 1014 cm−2, equivalent to a 1% As substitutional dopant concentration to a depth of ∼14 nm. Different substitutional As doses measured with MEIS were found to correlate closely with sheet resistance measurements, confirming that equating the substitutional As with the active As dose remains correct for these ultrashallow profiles, typically 10 nm deep.

Plasma doping ion implantation (PLAD) is becoming increasingly important for enabling the manufacture of advanced semiconductor devices. In this study, a VIISTA PLAD implanter was used to implant planar 300 mm Si wafers with As/7 keV from an arsine containing plasma with a total ion fluence of 1 × 1016 ions/cm2. The wafers then underwent a wet chemical clean and anneal to mimic a full industrial process flow. The effects of each process step were measured using cross-sectional TEM images, TEM/energy dispersive spectroscopy measurements, and medium energy ion scattering (MEIS). The PLAD implantation process was modeled using dynamic trim (TRIDYN), a dynamic, binary collision approximation model that accounted for the interactions between wafers and the ions and neutrals produced by the PLAD implanter. MEIS spectra were analyzed to extract elemental concentration depth profiles using POWERMEIS guided by the outputs of the TRIDYN model. The input fluxes of the TRIDYN model were calibrated such that the predicted TRIDYN and MEIS profiles were self-consistent. Combining the different analysis techniques and considering elemental concentrations alongside a TRIDYN model enabled magnitudes of ion and neutral fluxes of Si, As, and H to be proposed, and the relative importance of direct implantation and ion beam mixing during the PLAD implant to be revealed. This, in turn, led to proposals for the sources of the ion and neutral species, the importance of Si neutrals originating from the plasma chamber over those originating from the Si bulk in the “deposited” layer being of particular interest. Following the evolution of the as-implanted profiles through the wet clean and anneal steps gave insights into how the PLAD implant affected the results of the full process flow.

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.

The atomic layer deposition of titanium oxide TiO2 on ruthenium and oxidized ruthenium with titanium methoxide as metal precursor and H2O and O3 as oxidant was investigated by Rutherford backscattering (RBS) and time of flight secondary ion mass spectrometry (TOFSIMS). An ultra-thin layer of TiO2 deposited a priori with H2O plays the role of protection of Ru(Ox) substrates against etching by O3. Information about thin films (~3 nm) interfacial reactions, thickness and structure was brought by Medium Energy Ion Scattering Spectroscopy (MEIS) and X-ray absorption spectroscopy (XAS) measurements. The growth enhancements observed in the first stages of the deposition depends on the pre-treatment (pre-oxidation, H2O based interlayer thickness) of the Ru substrate. Thick films (~14 nm) were analyzed by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The as deposited TiO2 films are crystalline with rutile structure, as resulted from structural analyzes. However, the presence of small amounts of anatase was detected by soft X-ray absorption spectroscopy (XAS) and is strongly influenced by the surface pre-treatment of the Ru substrate. The electrical properties (equivalent oxide thickness and leakage current density) correlate with a different rutile/anatase ratio present in the films.

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.

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 0–6andnbsp;Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24–72andnbsp;h exposure to 250–750 μM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24andnbsp;h for all cell lines. Exposure to 750andnbsp;μM meloxicam for 24 h increased the fraction of cells in the radiosensitive G2/M cell cycle phase in D384 (18–27%) and U251 (17–41%) cells. 750 μM 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.

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. Mol Cancer Ther; 14(6); 1434–44. ©2015 AACR.

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.

In order to study the value of national health interview surveys for national and international research and policy activities, this paper examines the existence and content of recent and future health interview surveys in the 15 member states of the European Union (EU), Norway, Iceland and Switzerland. National health interview surveys are performed in most countries, but not in Greece (only regional surveys), Luxembourg, Ireland and Iceland (only multi-purpose surveys). The health interview surveys in the other 14 countries provide regular data on the main health topics. Of the 14 health topics that are examined in this inventory seven are measured in all countries. Questions on health status (e.g. self-assessed health, long-term physical disability, and height and weight) and medical consumption (e.g. consultations with the general practitioner, GP) are often included. Lifestyle topics are less often included, except smoking habits, information about which is sought in all countries. Topics like diet and drugs/narcotics are more often included in special surveys than in general health interview surveys. Despite differences in the content, frequency and methodology of national health interview surveys in different countries, these surveys are a valuable source of information on the health of Europeans.

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.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.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).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.

PURPOSE: This study was performed to determine the effects of a continuous exposure to paclitaxel (taxol) in combination with fractionated irradiation on cell proliferation and survival.METHODS AND MATERIALS: Human lung carcinoma cells (SW1573) were given a daily treatment with 3 Gy of x-rays during 5 days in the continuous presence of 5 nM taxol. The surviving fraction and the total number of cells were determined every 24 h before and immediately after irradiation.RESULTS: Irradiation with 5 x 3 Gy and 5 nM taxol cause approximately the same inhibition of cell proliferation. In combination these treatments have an additional effect and the cell population increases no further after the first 24 h. Whereas the cells become more resistant to taxol after the first 24 h with a minimum survival of 42%, taxol progressively reduces the population of surviving cells in combination with x-rays when the number of fractions increases, up to 25-fold relative to irradiation alone. The enhancement effect of 5 nM taxol is likely to be attributed to an inhibition of the repopulation during fractionated irradiation and not to an increased radiosensitivity. Only after treatment with 10 or 100 nM taxol for 24 h, which is attended with a high cytotoxicity, is moderate radiosensitization observed.CONCLUSION: Taxol, continuously present at a low concentration with little cytotoxicity, causes a progressive reduction of the surviving cell population in combination with fractionated irradiation, mainly by an inhibition of the repopulation of surviving cells between the dose fractions.

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 gamma-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 gamma-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 gamma-radiation. In both cell lines VPA caused enhancement of the radiation response after preincubation (DMF0.2 1.4 and 1.5) but not after postirradiation (DMF0.2 1.1 and 1.0). The combination of VPA and TMZ caused enhanced cytotoxicity (DMF0.2 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 (DMF0.2 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 gamma-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

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).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.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.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.

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.A model was designed that can predict radiation-drug interactions based on changes in clonogenic capacity and cell proliferation by radiation modifying agents.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.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.

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.

PURPOSE: Patients with a malignant glioma have a very poor prognosis. Cyclooxygenase-2 (COX-2) protein is regularly upregulated in gliomas and might be a potential therapeutic target. The effects of three selective COX-2 inhibitors were studied on three human glioma cell lines.MATERIALS AND METHODS: The selective COX-2 inhibitors NS-398, Celecoxib and Meloxicam and three human glioma cell lines (D384, U251 and U87) were used. Cell growth was assessed by a proliferation assay, the interaction with radiation (0 - 6 Gy) was studied using the clonogenic assay and cell cycle distribution was determined by FACS (fluorescence-activated cell sorting) analysis.RESULTS: All COX-2 inhibitors reduced proliferation of the glioma cell lines irrespective of their COX-2 expression level. Incubation with 200 microM NS-398 24 h before radiation enhanced radiation-induced cell death of D384 cells and 750 microM Meloxicam resulted in radiosensitization of D384 and U87 cells. No radiosensitization was observed with COX-2 inhibitor administration after radiotherapy. Treatment of D384 with NS-398 (200 microM) or Celecoxib (50 microM) and U87 with NS-398 (200 microM) after radiation resulted even in radioprotection.CONCLUSIONS: Effectiveness of COX-2 inhibitors on cell proliferation and radio-enhancement was independent of COX-2 protein expression. The sequence of COX-2 inhibitor addition and irradiation is very important.

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 of119 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.

A common feature of Glioblastoma Multiforme (GBM) is an augmented RTK/RAS/PI3K pathway signaling. This pathway has been shown to be involved in DNA repair by both NHEJ and HR in various tumor types and is thought thereby to interfere with its chemo- and radioresistance. Targeting this pathway to sensitize GBM to chemo- and radiotherapy seems to be an attractive therapeutic approach. Proliferation and clonogenic survival of U251, U87 and T98 GBM cells were assessed following exposure to, BKM120 (pan class I PI3K inhibitor), RAD001 (mTORC1 inhibitor), MK-2206 (AKT inhibitor) and/or MEK162 (ERK inhibitor) alone and in combination with either irradiation or Temozolomide (TMZ). Effectivity of each compound on its target pathway was assessed by westernblot. BEZ235 (PI3K/mTOR/DNA-PK inhibitor) was used as a positive control for radiosensitization. Our data show BKM120 and MK-2206 to effectively inhibit AKT phosphorylation at 1 uM and leads to growth inhibition in all tested cell lines. However, these compounds are not toxic on clonogenic cell survival and do not sensitize the cells to TMZ or irradiation. Increasing the dose to 5 uM and 10 uM for BKM120 and MK-2206 respectively an antagonistic effect was found in the combination with TMZ and irradiation. Effectively inhibiting mTOR, the downstream effector of AKT, by 100 nM RAD001 also does not radiosensitize GBM cells. Inhibiting the parallel ERK-pathway by 1 uM MEK162, dephosphorylates ERK1/2 and leads to a slight increase in AKT phosphorylation. Inhibiting both pathways with BKM120 and MEK162 resulted in a synergistic growth inhibition of U251 cells however no radiosensitizing effect was found in a clonogenic assay. In conclusion, selective targeting of key components in the AKT- and/or ERK signaling pathway effectively inhibits proliferation of GBM cells yet does not sensitize them to either TMZ or radiotherapy, indicating that GBM does not depend on the RTK/RAS/PI3K pathway for its DNA-repair. Citation Format: Ravi Narayan, Rogier Dik, Jaap van den Berg, Lukas J.A. Stalpers, Brigitta G. Baumert, Ben J. Slotman, Peter Sminia. AKT- and ERK signaling inhibition does not sensitize GBM cells to chemo-/radiation in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4914. doi:10.1158/1538-7445.AM2014-4914

Purpose: To investigate the effect of temozolomide, a 3-methyl derivative of mitozolomide in combination with X-rays in human glioma-derived cell lines. Methods and Materials: Glioma cell lines D384 and U251 were treated with temozolomide for various periods of time in combinations with X-rays. Temozolomide administration was repeated every 24 h for exposures up to 96 h. Cytotoxicity was determined with a clonogenic assay. Results: Incubation of D384 cells with temozolomide during 24 h prior to or following irradiation results in a moderate enhancement of the cytotoxicity. Prolonged treatment with temozolomide, i.e., 48–96 h before X-rays, causes a stronger potentiation. In contrast, no enhancement is observed in irradiated U251 cells in combination with 24–96 h temozolomide treatment. In addition to single-dose irradiation, we investigated the effect of temozolomide in D384 cells with concomitant fractionated irradiation. A 96-h exposure to temozolomide with simultaneous doses of 2 Gy X-rays at 24-h intervals, causes a significant further reduction in cell survival as compared to fractionated irradiation only. Conclusion: Depending on the cell line, treatment of glioma cells with temozolomide and X-rays can have either an additional effect or potentiate cell killing.

Introduction: Over 50% of all cancer patients receive radiotherapy during their treatment. Recent studies demonstrate that combining radiotherapy with angiostatic drugs may have a beneficial effect on tumor growth inhibition in vivo. However, efficacy depends on the type of drug, the dose and the scheduling. We adapted the chick chorioallantoic membrane (CAM) assay to allow rapid analysis of the effects of radiotherapy in combination with angiostatic drugs on in vivo angiogenesis. Using this model, we studied the effects of different treatment schedules with sunitinib on tumors grafted onto the CAM. Methods: The CAMs of fertilized chicken eggs were treated daily with sunitinib (50 μL of 10 μM) starting at embryonic day of development (EDD) 6. Irradiation was given using a cobalt-60 source. On EDD10 the CAM vasculature was imaged and analyzed using specific software (HetCAM, DCI labs). For the in vivo tumor model, 5x106 HT29 or D384 cells were grafted on the CAM on EDD6. From EDD10 onwards, combination therapy was applied to the tumors using different treatment schedules. Tumor growth was measured daily until EDD17 after which tumor were dissected for further analysis. Results: Single dose (SD) radiotherapy (4 Gy) on EDD6 induces an almost 50% reduction in the vessel length and the number of branchpoints. Within 3 days these vascular parameters normalize. Sunitinib treatment for 2 and 4 days results in significant reduction of the vascular parameters. Applying 4 Gy after sunitinib treatment has an additive effect on the vasculature. In the tumor grafts, SD radiotherapy (4 Gy) decreased tumor volume and weight significantly four days after irradiation which was mainly caused by impaired tumor cell growth rather than by vascular defects. Combining radiotherapy (SD of 4Gy) with sunitinib treatment (4x 50 uL of 0.8uM) only had a beneficial effect when irradiation was given within the first 3 days of sunitinib treatment. Furthermore, while SD irradiation with 2 Gy alone did not affect tumor growth, a beneficial effect was observed when 2 Gy irradiation was given at the start of sunitinib treatment. Conclusion: The effect of combining radiotherapy with sunitinib on in vivo angiogenesis and tumor growth depends on the treatment schedule. Optimal scheduling allows a reduction in radiation dose. Preclinical analysis of optimal dose/scheduling is essential for the design of clinical trials and for translation of combination therapy to the clinic. Citation Format: Esther A. Kleibeuker, Kitty C. Castricum, Jaap van den Berg, Richard Honeywell, Arjan W. Griffioen, Ben J. Slotman, Henk M. Verheul, Victor L. Thijssen. Improving the radio-sensitizing effect of sunitinib by drug-specific scheduling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2013-1586

Purpose: To investigate the role of the cyclic adenosie 3′:5′-monophosphate (AMP) signal transduction pathway in heat-induced cell death and the development of thermotolerance. Methods and Materials: Reuber H35 rat hepatoma cells were heated after preincubation with various compounds known to modulate the cyclic AMP signal transduction pathway. Cell survival was determining by colony-forming ability. Results: Preincubation of H35 cells with forskolin, a stimulator of adenylate cyclase, in combination with IBMX (3-isobutyl-1-methylxanthine), an inhibitor of cyclic AMP phosphodiesterase, results in thermosensitization. Similar results are obtained with various cyclic AMP analogs. Maximum thermosensitization occurs with 0.5 mM dibutyryl cyclic AMP (DBcAMP) after a preincubation period of 5 h and heating in the presence of the drug. The same relative degree of thermosensitization is found with 8-Cl-cAMP, but a 10-fold lower concentration Thermosensitization by DBcAMP is prevented by H89, a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Without additional cyclic AMP-inducing factors, H89 induces thermoprotection. Node of the drug treatments are cytotoxic at 37°C. DBcAMP does not affect the development of heat-induced thermotolerance but it reduces its experession to an extent similarly found in the observed thermosensitization in nonthermotolerant cells. Conclusion: The results strongly indicate that the cyclic AMP signal transduction pathway is involved in the process of heat-induced cell death. DBcAMP reduces the expression of thermotolerance, but does not affect its induction.

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