Dose Enhancement Ratio Research Articles

In vitro and in silico study of biological effects on cancer cells in the presence of metallic materials during radiotherapy.

X-ray therapy aims to eliminate tumours while minimizing side effects. Intense mucositis is sometimes induced when irradiating the oral cavity with a dental metal crown (DMC). However, the underlying mechanisms of such inducing radiosensitization by DMC remain uncertain. This study explored the radiosensitizing mechanisms around DMCs in an interdisciplinary approach with cell experiments and Monte Carlo simulation with the PHITS code. Clonogenic survival and nuclear 53BP1 foci of a cell line derived from cervical cancer cells (HeLa cells) were measured post-irradiation with therapeutic X-rays near high-Z materials such as Pb or Au plates, and the experimental sensitizer enhancement ratio (SER) was obtained. Meanwhile, the dose enhancement ratio (DER) and relative biological effectiveness for DNA damage yields were calculated using the PHITS code, by considering the corresponding experimental condition. The experiments show the experimental SER values for cell survival and 53BP1 foci near metals are 1.2-1.4, which agrees well with the calculated DER values. These suggest that the radiosensitizing effects near metal are predominantly attributed to the dose increase. In addition, as a preclinical evaluation, the spatial distributions of DER near DMC are calculated using Computed Tomography Digital Imaging and Communications in Medicine (CT-DICOM) data and a simple tooth model. As a result, the DER values evaluated using the CT-DICOM data were lower than those from a simple tooth model. These findings highlight the challenge of evaluating radiosensitizing effects near DMCs using Digital Imaging and Communications in Medicine (DICOM) images due to volume-averaging effects and emphasize the need for a high-resolution (<1mm) dose assessment method unaffected by these effects.

Equivalent uniform aerobic dose in radiotherapy for hypoxic tumors

Objective. Equivalent uniform aerobic dose (EUAD) is proposed for comparison of integrated cell survival in tumors with different distributions of hypoxia and radiation dose. Approach. The EUAD assumes that for any non-uniform distributions of radiation dose and oxygen enhancement ratio (OER) within a tumor, there is a uniform distribution of radiation dose under hypothetical aerobic conditions with OER = 1 that produces equal integrated survival of clonogenic cells. This definition of EUAD has several advantages. First, the EUAD allows one to compare survival of clonogenic cells in tumors with intra-tumor and inter-tumor variation of radio sensitivity due to hypoxia because the cell survival is recomputed under the same benchmark oxygen level (OER = 1). Second, the EUAD for homogeneously oxygenated tumors is equal to the concept of equivalent uniform dose. Main results. We computed the EUAD using radiotherapy dose and the OER derived from the 18F-Fluoromisonidazole PET (18F-FMISO PET) images of hypoxia in patients with glioblastoma, the most common and aggressive type of primary malignant brain tumor. The 18F-FMISO PET images include a distribution of SUV (Standardized Uptake Value); therefore, the SUV is converted to partial oxygen pressure (pO2) and then to the OER. The prognostic value of EUAD in radiotherapy for hypoxic tumors is demonstrated using correlation between EUAD and overall survival (OS) in radiotherapy for glioblastoma. The correction to the EUAD for the absolute hypoxic volume that traceable to the tumor control probability improves the correlation with OS. Significance. While the analysis proposed in this research is based on the 18F-FMISO PET images for glioblastoma, the EUAD is a universal radiobiological concept and is not associated with any specific cancer or any specific PET or MRI biomarker of hypoxia. Therefore, this research can be generalized to other cancers, for example stage III lung cancer, and to other hypoxia biomarkers.

Monte Carlo study on nanogold dose enhancement for breast radiotherapy using an ICRP female computational phantom

Due to distinct photoelectric absorption properties and physiochemical features, high-atomic-element nanomaterials are frequently used as radiosensitizer for radiotherapy, such as gold nanoparticles. which in-turn can increase radiation dose to tumours while minimizing exposure to normal tissues. The aim of this study is to investigate the dose enhancement due to the presence of nanogold particles mixed with breast tissue using a real radiotherapy plan with two photon energies, with and without flattening filter. A fully validated linac head was used to model a complete field-in-field (FIF) radiotherapy breast plan using 4 and 6 MV with a flattening filter (FF) and flattening filter-free (FFF) using BEAMnrc/EGSnrc MC code. The dose distributions were simulated in a female ICRP voxel computational phantom. The dose enhancement ratio (DER) and flattening filter-free enhancement ratio (FFFER) were calculated for a range of nanogold concentrations. The results showed that the DER increased with increasing nanogold concentration. The maximum DER was achieved when FFF was used and the increase was higher for 4 MV than 6 MV. The FFFER was higher for the 4 MV beam than 6 MV when 40 mg/ml nanogold was used. The combination of higher nanogold concentrations, lower photon energies, and FFF resulted in a higher dose enhancement. The implementation of nanogold particles in breast tissue enhanced the dose deposition, especially with FFF, as presented in this study. The results would improve nanogold-enhanced breast radiotherapy and dose delivery with an increase in patient throughput.

MRI Detection and Therapeutic Enhancement of Ferumoxytol Internalization in Glioblastoma Cells.

Recently, the FDA-approved iron oxide nanoparticle, ferumoxytol, has been found to enhance the efficacy of pharmacological ascorbate (AscH-) in treating glioblastoma, as AscH- reduces the Fe3+ sites in the nanoparticle core. Given the iron oxidation state specificity of T2* relaxation mapping, this study aims to investigate the ability of T2* relaxation to monitor the reduction of ferumoxytol by AscH- with respect to its in vitro therapeutic enhancement. This study employed an in vitro glioblastoma MRI model system to investigate the chemical interaction of ferumoxytol with T2* mapping. Lipofectamine was utilized to facilitate ferumoxytol internalization and assess intracellular versus extracellular chemistry. In vitro T2* mapping successfully detected an AscH--mediated reduction of ferumoxytol (25.6 ms versus 2.8 ms for FMX alone). The T2* relaxation technique identified the release of Fe2+ from ferumoxytol by AscH- in glioblastoma cells. However, the high iron content of ferumoxytol limited T2* ability to differentiate between the external and internal reduction of ferumoxytol by AscH- (ΔT2* = +839% for external FMX and +1112% for internal FMX reduction). Notably, the internalization of ferumoxytol significantly enhances its ability to promote AscH- toxicity (dose enhancement ratio for extracellular FMX = 1.16 versus 1.54 for intracellular FMX). These data provide valuable insights into the MR-based nanotheranostic application of ferumoxytol and AscH- therapy for glioblastoma management. Future developmental efforts, such as FMX surface modifications, may be warranted to enhance this approach further.

Targeting Tri-Methyl-Histone H3 (Lys27) in Rectal Cancer: A Novel Strategy to Enhance Radiosensitivity and Predict Response to Neoadjuvant Therapy

Tri-Methyl-Histone H3 (Lys27) is a modification of histone H3 involved in gene regulation and chromatin organization. It plays a crucial role in cell response to ionizing radiation and affects DNA damage repair, with altered levels being linked to decreased radiation sensitivity. Our hypothesis is that high levels of Tri-Methyl-Histone H3 (Lys27) in rectal tumors can predict response to neoadjuvant chemoradiation, and targeting SETD2, the histone methyltransferase responsible for adding the tri-methyl group to Lys27 of histone H3, may enhance radiosensitivity in rectal cancer cells. Biopsy samples from 25 patients with locally advanced rectal adenocarcinoma (Stage II-III) were obtained for analysis. Immunohistochemistry (IHC) was used to evaluate Tri-Methyl-Histone H3 (Lys27) expression levels in the tumor biopsies. The staining intensity was scored semi-quantitatively. Samples with 100% positive staining were grouped as "Tri-Methyl-High," and all others were grouped as "Tri-Methyl-Low." Fisher's exact test was used to analyze the data to determine the correlation between Tri-Methyl-Histone H3 (Lys27) levels and response to neoadjuvant chemoradiation. The primary endpoint was either a sustained clinical complete response (cCR) at one year or a pathological complete response (pCR). To further understand the role of Tri-Methyl-Histone H3 (Lys27) in response to ionizing radiation (IR), clonogenic assays, γH2aX foci staining, and western blot analysis of DNA damage response proteins and histones were performed on two established human rectal cancer cell lines following either RNA inhibition (RNAi) or drug targeting of SETD2. Twenty-five patients with rectal cancer had pre-treatment biopsies analyzed for Tri-Methyl-Histone H3 (Lys27) levels, 11 were classified as Tri-Methyl-High and 14 as Tri-Methyl-Low. The groups were well balanced in terms of age, sex, clinical T/N stage, and neoadjuvant treatment approach. The overall combined complete response (cCR/pCR) rate was 36%, while the rate for Tri-Methyl-High patients was 9% (1/11) compared to 57% (8/14) in Tri-Methyl-Low patients (p = 0.03). After treating two colorectal cancer cell lines (SW837 and HCT116) with IR (5 Gy) in vitro, Tri-Methyl-Histone H3 (Lys27) levels increased 3-fold. SETD2 RNAi reduced Tri-Methyl-Histone H3 (Lys27) and increased cell death when combined with IR. In vitro treatment with a novel SETD2 inhibitor, EZM0414, led to a 2-fold increase in DNA damage following IR (5 Gy) as measured by γH2aX foci staining. EZM0414 treatment showed a 37% improvement in the dose enhancement ratio. Our study uncovers a new biomarker, Tri-Methyl-Histone H3 (Lys27), that could be used to predict response to neoadjuvant chemoradiation in rectal cancer patients. Our preclinical data indicates that targeting SETD2 to reduce Tri-Methyl-Histone H3 (Lys27) mediated DNA repair could improve the efficacy of radiation therapy for rectal cancer patients.

Cell-type specific anti-cancerous effects of nitro-oleic acid and its combination with gamma irradiation.

Nitro-fatty acids (NFAs) are endogenous lipid mediators capable of post-translational modifications of selected regulatory proteins. Here, we investigated the anti-cancerous effects of nitro-oleic acid (NO2OA) and its combination with gamma irradiation on different cancer cell lines. The effects of NO2OA on cell death, cell cycle distribution, or expression of p21 and cyclin D1 proteins were analyzed in cancer (A-549, HT-29 and FaDu) or normal cell lines (HGF, HFF-1). Dose enhancement ratio at 50 % survival fraction (DERIC50) was calculated for samples pre-treated with NO2OA followed by gamma irradiation. NO2OA suppressed viability and induced apoptotic cell death. These effects were cell line specific but not in general selective for cancer cells. HT-29 cell line exerted higher sensitivity toward NO2OA treatment among cancer cell lines tested: induction of cell cycle arrest in the G2/M phase was associated with an increase in p21 and a decrease in cyclin D1 expression. Pre-treatment of HT-29 cells with NO2OA prior irradiation showed a significantly increased DERIC50, demonstrating radiosensitizing effects. In conclusion, NO2OA exhibited potential for combined chemoradiotherapy. Our results encourage the development of new NFAs with improved features for cancer chemoradiation.

Depth Dose Enhancement in Orthovoltage Nanoparticle-Enhanced Radiotherapy: A Monte Carlo Phantom Study.

This study was to examine the depth dose enhancement in orthovoltage nanoparticle-enhanced radiotherapy for skin treatment by investigating the impact of various photon beam energies, nanoparticle materials, and nanoparticle concentrations. A water phantom was utilized, and different nanoparticle materials (gold, platinum, iodine, silver, iron oxide) were added to determine the depth doses through Monte Carlo simulation. The clinical 105 kVp and 220 kVp photon beams were used to compute the depth doses of the phantom at different nanoparticle concentrations (ranging from 3 mg/mL to 40 mg/mL). The dose enhancement ratio (DER), which represents the ratio of the dose with nanoparticles to the dose without nanoparticles at the same depth in the phantom, was calculated to determine the dose enhancement. The study found that gold nanoparticles outperformed the other nanoparticle materials, with a maximum DER value of 3.77 at a concentration of 40 mg/mL. Iron oxide nanoparticles exhibited the lowest DER value, equal to 1, when compared to other nanoparticles. Additionally, the DER value increased with higher nanoparticle concentrations and lower photon beam energy. It is concluded in this study that gold nanoparticles are the most effective in enhancing the depth dose in orthovoltage nanoparticle-enhanced skin therapy. Furthermore, the results suggest that increasing nanoparticle concentration and decreasing photon beam energy lead to increased dose enhancement.

Abstract 2839: Methionine restriction radiosensitizes KRAS mutant rectal cancer

Abstract Purpose/Objective(s): Radiation therapy (RT) is the standard of care in patients with locally advanced rectal cancer, but not all patients respond equally to RT. KRAS-mutated (KRASmut) cancer cells are highly radioresistance and require higher levels of methionine, an essential amino acid, compared to KRAS wildtype (KRASwt) and normal cells, and diet restriction of methionine has been shown to potentiate the effects of radiation in vivo. We hypothesized that methionine restriction (MR) could radiosensitize KRASmut rectal cancer cells. Materials/Methods: HCT116 KRASG13D/+ and SW48 KRASwt colorectal carcinoma isogenic cell lines and their isogenic derivative cell lines HCT 116 KRAS+/- and SW48 KRASG13D were obtained from Horizon Discovery Ltd (Cambridge, UK). Radiation clonogenic assays, immunofluorescence, and western blot analysis of key DNA damage response proteins were performed in the KRASmut vs. KRASwt colorectal cells treated with ionizing radiation (IR, 2-6 Gy) at increasing time points following pre-treatment with methionine control (MC) (200 µM) or MR (40-fold reduction in methionine, 5 µM) media for 48 hours. One hundred thousand luciferase-expressing KRASwt or KRASmut SW48 cells were implanted orthotopically in the rectum of athymic nude mice. After confirmation of tumor formation at one-week post-injection, the mouse diet was changed to either an MC (0.84% methionine w/w) or MR (0.12% methionine w/w) diet for a total of two weeks. Rectal tumors were treated on an Xstrahl small animal radiation research platform (SARRP) to a dose of 25 Gy in 5 daily fractions (1 cm x 1 cm collimator, isocenter at anal verge) during the second week of diet modification. Tumor growth was measured via bioluminescence on IVIS Lumina every two weeks for four weeks post-radiation. Results: MR significantly increased radiosensitization compared to MC in both HCT116 and SW48 cells in vitro. KRASmut cells showed a 20-30% increase in radiation in the dose enhancement ratio compared to the KRASwt cells. The protein expression and nuclear foci per cell of the DNA damage marker γH2AX were significantly higher in the KRASmut cells compared to KRASwt cells following 5 Gy of RT at 6 hours. Protein expression of homologous recombination pathway proteins RAD51 and the CtBP interacting protein (CtIP) were higher at baseline in the KRASmut cells than KRASwt. MR substantially reduced the expression of these proteins following RT. Mice harboring SW48 KRASmut rectal tumors treated with 5 Gy x 5 RT and MR showed a significant increase in the complete response rate compared to MC (80% vs. 10%, p=0.02). In the mice harboring SW48 KRASwt rectal tumors, there was no significant difference in response rate between MR vs. MC (40% vs. 30%, p=NS). Conclusion: Our study indicates a novel strategy to radiosensitize resistant KRAS-mutated rectal cancer through MR. Studies testing the mechanism of KRAS-dependent methionine-mediated DNA repair is ongoing. Citation Format: Adam R. Wolfe, Sarita Garg, Stetson Van Matre, Robin Eluvathingal, Oscar Zuniga, Henrique Rodrigues, Nükhet Aykin-Burns, Isabelle Racine Miousse. Methionine restriction radiosensitizes KRAS mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2839.

Discovery of a Novel Driver of Radioresistance in Pancreatic Cancer (PC) Using Genome-Wide RNA-Seq

<h3>Purpose/Objective(s)</h3> Discovery of genetic drivers of radioresistance in PC is critical for developing novel therapeutic strategies to combine with radiation in this radioresistant disease. In this study, we used genome-wide RNA-seq to identify genes upregulated in radioresistant (RR) generated PC cell lines and discovered the Inhibitor of DNA Binding 1 (ID1) gene as a promising radiosensitizing target. <h3>Materials/Methods</h3> RR PC cell lines MIA PaCa-2 and PANC-1 were generated by delivering daily ionizing irradiation (IR) (2 Gy/day) <i>in vitro</i> over two weeks (total 20 Gy) followed by standard clonogenic assays to calculate the dose enhancement ratio (DER) following one week from the end of IR. Parental and RR cell lines were submitted for RNA-seq analysis to identify differentially expressed genes. The Limma R package was used to calculate differential expression among genes. Log2 fold change values were calculated for each sample compared to control. Genes with an absolute fold change > 1 were considered significant. Knockdown of target genes via small interfering RNA (siRNA) was confirmed through immunoblotting. Luciferase expressing RR PANC-1 and MIA PaCa-2 with or without shRNA stable knockdown of ID1 were implanted orthotopically in the pancreas of athymic nude mice, and tumors were treated with external beam radiation using a Small Animal Radiation Research Platform to a dose of 20 Gy in 5 daily fractions. Tumor growth was measured weekly via bioluminescence. <h3>Results</h3> Following exposure to two weeks of 2 Gy daily IR <i>in vitro</i>, the two PC cell lines showed significantly greater clonogenic cell survival than parental cell lines (DER 0.4 and 0.5), indicating enhanced RR in these cells. RNA-seq analysis comparing parental and RR cell lines found upregulated seven genes (TNS4, ZDHHC8P1, APLNR, AQP3, SPP1, ID1, ID2) and seven genes downregulated (PTX3, ITGB2, EPS8L1, ALDH1L2, KCNT2, ARHGAP9, IFI16) in both RR cell lines. Western blotting confirmed either increased or decreased expression of these 14 genes in the RR cell lines compared to their parental. siRNA knockdown of each of the seven most significant upregulated genes in the RR MIA PaCa-2 and PANC-1 cells revealed ID1 as the most potent radiosensitizer in the RR cell lines (DER 1.5 and 1.6, respectively). ID1 protein expression was higher in 6 different human PC cell lines than both non-malignant pancreatic and intestinal cell lines. Orthotopic RR tumors with stable knockdown of ID1 showed significantly decreased tumor growth following <i>in vivo IR</i> than the wild-type ID1 tumors (2.5-fold decrease, p<0.001). After the last dose of IR, expression of the DNA damage marker yH2aX in tumors was significantly higher in the ID1 knockdown tumors compared to controls indicating reduced DNA repair in tumors deficient in ID1. <h3>Conclusion</h3> Our analysis indicates a novel role of ID1 in PC radioresistance. Targeting ID1 prior to IR is a promising strategy to overcome the RR of PC, and pre-clinical studies using ID1 inhibitors are ongoing.

JNC-1043, a Novel Podophyllotoxin Derivative, Exerts Anticancer Drug and Radiosensitizer Effects in Colorectal Cancer Cells.

The objective of this study was to determine whether (5S)-5-(4-benzyloxy-3,5-dimethoxy-phenyl)-5,9-dihydro-8H-furo [3’,4’:6,7] naphtho [2,3-d] [1,3]dioxol-6-one (JNC-1043), which is a novel chemical derivative of β-apopicropodophyllin, acts as a novel potential anticancer reagent and radiosensitizer in colorectal cancer (CRC) cells. Firstly, we used MTT assays to assess whether JNC-1043 could inhibit the cell proliferation of HCT116 and DLD-1 cells. The IC50 values of these cell lines were calculated as 114.5 and 157 nM, respectively, at 72 h of treatment. Using doses approximating the IC50 values, we tested whether JNC-1043 had a radiosensitizing effect in the CRC cell lines. Clonogenic assays revealed that the dose-enhancement ratios (DER) of HCT116 and DLD-1 cells were 1.53 and 1.25, respectively. Cell-counting assays showed that the combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. Treatment with JNC-1043 or IR alone induced cell death by 50~60%, whereas the combination of JNC-1043 and IR increased this cell death by more than 20~30%. Annexin V-propidium iodide assays showed that the combination of JNC-1043 and IR increased apoptosis by more 30~40% compared to that induced by JNC-1043 or IR alone. DCFDA- and MitoSOX-based assays revealed that mitochondrial ROS production was enhanced by the combination of JNC-1043 and IR. Finally, we found that suppression of ROS by N-acetylcysteine (NAC) blocked the apoptotic cell death induced by the combination of JNC-1043 and IR. The xenograft model also indicated that the combination of JNC-1043 and IR increased apoptotic cell death in tumor mass. These results collectively suggest that JNC-1043 acts as a radiosensitizer and exerts anticancer effects against CRC cells by promoting apoptosis mediated by mitochondrial ROS.

Quantifying Radiosensitization of PSMA-Targeted Gold Nanoparticles on Prostate Cancer Cells at Megavoltage Radiation Energies by Monte Carlo Simulation and Local Effect Model.

Active targeting gold nanoparticles (AuNPs) are a very promising avenue for cancer treatment with many publications on AuNP mediated radiosensitization at kilovoltage (kV) photon energies. However, uncertainty on the effectiveness of AuNPs under clinically relevant megavoltage (MV) radiation energies hinders the clinical translation of AuNP-assisted radiation therapy (RT) paradigm. The aim of this study was to investigate radiosensitization mediated by PSMA-targeted AuNPs irradiated by a 6 MV radiation beam at different depths to explore feasibility of AuNP-assisted prostate cancer RT under clinically relevant conditions. PSMA-targeted AuNPs (PSMA-AuNPs) were synthesized by conjugating PSMA antibodies onto PEGylated AuNPs through EDC/NHS chemistry. Confocal fluorescence microscopy was used to verify the active targeting of the developed PSMA-AuNPs. Transmission electron microscopy (TEM) was used to demonstrate the intracellular biodistribution of PSMA-AuNPs. LNCaP prostate cancer cells treated with PSMA-AuNPs were irradiated on a Varian 6 MV LINAC under varying depths (2.5 cm, 10 cm, 20 cm, 30 cm) of solid water. Clonogenic assays were carried out to determine the in vitro cell survival fractions. A Monte Carlo (MC) model developed on TOPAS platform was then employed to determine the nano-scale radial dose distribution around AuNPs, which was subsequently used to predict the radiation dose response of LNCaP cells treated with AuNPs. Two different cell models, with AuNPs located within the whole cell or only in the cytoplasm, were used to assess how the intracellular PSMA-AuNP biodistribution impacts the prostate cancer radiosensitization. Then, MC-based microdosimetry was combined with the local effect model (LEM) to calculate cell survival fraction, which was benchmarked against the in vitro clonogenic assays at different depths. In vitro clonogenic assay of LNCaP cells demonstrated the depth dependence of AuNP radiosensitization under clinical megavoltage beams, with sensitization enhancement ratio (SER) of 1.14 ± 0.03 and 1.55 ± 0.05 at 2.5 cm depth and 30 cm depth, respectively. The MC microdosimetry model showed the elevated percent of low-energy photons in the MV beams at greater depth, consequently resulting in increased dose enhancement ratio (DER) of AuNPs with depth. The AuNP-induced DER reached ~5.7 and ~8.1 at depths of 2.5 cm and 30 cm, respectively. Microdosimetry based LEM accurately predicted the cell survival under 6 MV beams at different depths, for the cell model with AuNPs placed only in the cell cytoplasm. TEM results demonstrated the distribution of PSMA-AuNPs in the cytoplasm, confirming the accuracy of MC microdosimetry based LEM with modelled AuNPs distributed within the cytoplasm. We conclude that AuNP radiosensitization can be achieved under megavoltage clinical radiotherapy energies with a dependence on tumor depth. Furthermore, the combination of Monte Carlo microdosimetry and LEM will be a valuable tool to assist with developing AuNP-aided radiotherapy paradigm and drive clinical translation.

Dosimetric Impact on the Flattening Filter and Addition of Gold Nanoparticles in Radiotherapy: A Monte Carlo Study on Depth Dose Using the 6 and 10 MV FFF Photon Beams.

Purpose: This phantom study investigated through Monte Carlo simulation how the dose enhancement varied with depth, when gold nanoparticles (NPs) were added using the flattening filter-free (FFF) photon beams in gold NP-enhanced radiotherapy. Method: A phantom with materials varying from pure water to a mixture of water and gold NPs at different concentrations (3–40 mg/mL) were irradiated by the 6 and 10 MV flattening filter (FF) and FFF photon beams. Monte Carlo simulations were carried out to determine the depth doses along the central beam axis of the phantom up to a depth of 40 cm. The dose enhancement ratio (DER) and FFF enhancement ratio (FFFER) were calculated based on the Monte Carlo results. Results: The DER values were found decreased with an increase of depth and increase of NP concentration in the phantom. For the maximum NP concentration of 40 mg/mL, the DER values decreased 6.9, 12, 4.6 and 7.2% at a phantom depth from 2 to 40 cm, using the 6 MV FF, 6 MV FFF, 10 MV FF and 10 MV FFF photon beams, respectively. The maximum DER values for the 6 MV beams were 1.08 (FF) and 1.14 (FFF), while those for the 10 MV beams were 1.04 (FF) and 1.07 (FFF). When the FF was removed from the linear accelerator head, the FFFER showed a more significant increase of dose enhancement for the 6 MV beams (1.057) than the 10 MV (1.031). Conclusion: From the DER and FFFER values based on the Monte Carlo results, it is concluded that the dose enhancement with depth was dependent on the NP and beam variables, namely, NP concentration, presence of FF in the beam and beam energy. Dose enhancement was more significant when using the lower photon beam energy (i.e., 6 MV), FFF photon beam and higher NP concentration in the study.

Evaluation of Dosimetric Effect of Bone Scatter on Nanoparticle-Enhanced Orthovoltage Radiotherapy: A Monte Carlo Phantom Study.

In nanoparticle (NP)-enhanced orthovoltage radiotherapy, bone scatter affected dose enhancement at the skin lesion in areas such as the forehead, chest wall, and knee. Since each of these treatment sites have a bone, such as the frontal bone, rib, or patella, underneath the skin lesion and this bone is not considered in dose delivery calculations, uncertainty arises in the evaluation of dose enhancement with the addition of NPs in radiotherapy. To investigate the impact of neglecting the effect of bone scatter, Monte Carlo simulations based on heterogeneous phantoms were carried out to determine and compare the dose enhancement ratio (DER), when a bone was and was not present underneath the skin lesion. For skin lesions with added NPs, Monte Carlo simulations were used to calculate the DER values using different elemental NPs (gold, platinum, silver, iodine, as well as iron oxide), in varying NP concentrations (3–40 mg/mL), at two different photon beam energies (105 and 220 kVp). It was found that DER values at the skin lesion increased with the presence of bone when there was a higher atomic number of NPs, a higher NP concentration, and a lower photon beam energy. When comparing DER values with and without bone, using the same NP elements, NP concentration, and beam energy, differences were found in the range 0.04–3.55%, and a higher difference was found when the NP concentration increased. By considering the uncertainty in the DER calculation, the effect of bone scatter became significant to the dose enhancement (>2%) when the NP concentration was higher than 18 mg/mL. This resulted in an underestimation of dose enhancement at the skin lesion, when the bone underneath the tumour was neglected during orthovoltage radiotherapy.

Outcomes following transarterial radioembolization with 90Y and nanoparticles loaded resin microspheres

90Y bremsstrahlung Single-Photon Emission Tomography (SPECT) imaging is employed to check the possibility of extrahepatic uptake and the quantification of delivered dose in Transarterial Radioembolization (TARE). 90Y bremsstrahlung SPECT imaging is challenging due to the nature of bremsstrahlung photons. We reported a Monte Carlo study using the resin microspheres loaded with 90Y and Nanoparticles (NPs) in the TARE. By injection of Bismuth (Bi) and Europium (Eu) NPs into the resin microspheres, the sensitivity and the contrast to noise ratio increased for the bremsstrahlung planar images. The highest signal to background ratio was observed in the characteristic X-ray planar images taken with the energy window at the Kα1 ± 10 keV when Eu NPs were incorporated into the microsphere. The dose enhancement ratio decreased dramatically at NP concentrations >2.4 M. Incorporating NPs into the resin microspheres improves the quality of post-treatment images and establishes a standardized imaging protocol for post-treatment imaging by characteristic X-rays.

Abstract 3312: A CRISPR-Cas9 screen in meningioma reveals GCN2/ATF4 is a novel target that enhances the response to radiation

Abstract Purpose: Meningioma is a common primary brain tumor in adults and accounts for ~ 36% of all adult brain tumors. Therapy for patients with meningioma is individualized and comprised of a combination of surgery and/or radiotherapy. Chemotherapy is not effective and there is no approved targeted treatment. Therefore, novel therapeutic approaches are warranted. We used CRISPR screening-based technology to uncover novel mechanisms of radiation resistance that may be therapeutically relevant in meningioma. Methods: A patient-derived meningioma cell line with TRAF7/KLF4 mutations (Tm77) was transduced with a Human Kinome CRISPR KnockOut (KO) library containing 3,052 RNA guides (sgRNA) targeting 763 kinase genes involved in cancer therapy. Following radiation, cells were collected (400x coverage) and analyzed using next-generation sequencing (NGS). MAGeCK analysis was used to evaluate sgRNA enrichment and depletion. Radiation survival was determined by clonogenic analysis. Kinase activity and downstream signaling were assessed by phospho-blot analysis. Cell cycle analysis was performed by flow cytometry and DNA damage was assessed by gH2AX immunofluorescence. Results: Analysis of NGS results shows that after radiation ATM and DNA-PK sgRNAs were depleted (p&amp;lt;0.001; FDR&amp;lt;0.001), suggesting that the inhibition of either induces radiosensitivity. These genes, together with ATR, are involved in the DNA damage response induced by external stress such as radiation. ATM (AZD1930) and ATR (AZD6738) inhibitors were used to test the screening results. While AZD1390 reduces ATM signaling, gH2AX foci, and radiation survival with a dose enhancement ratio (DER) at 2Gy of 2; AZD6738 did not induce any changes in signaling, gH2AX foci, or radiosensitivity. These results indicate that inhibition of ATM can regulate radiation response in meningioma and thus validated the screening results. The CRISPR-Cas9 KO screen also identified a potential novel druggable target, GCN2. In the NGS analysis, GCN2 sgRNAs were enriched (p&amp;lt;0.001; FDR&amp;lt;0.005), and in agreement with this finding GCN2-KO enhanced radiation survival with a DER at 2Gy of 1.4. In contrast, halofuginone, a natural product that activates GCN2 reduced radiation survival in Tm77 with a DER at 2Gy of 1.25 through activation of ATF4 and cell cycle arrest. These effects were not observed in GCN2-KO cell lines. In addition, the proteasome inhibitor, bortezomib, also activates ATF4 signaling, causes cell cycle arrest, and radiosensitization with a DER at 2Gy of 1.3. Conclusion: CRISPR-Cas9 KO screening provides a novel and unbiased approach to identify cellular targets that modify the response to radiation. These results suggests that the activation of ATF4 radiosensitizes meningioma. The evidence provided in this study supports further basic and preclinical research on approaches for activating GCN2 in combination with radiation therapy in meningioma. Citation Format: Marta Baro, Murat Gunel, Jennifer Moliterno, Joseph N. Contessa. A CRISPR-Cas9 screen in meningioma reveals GCN2/ATF4 is a novel target that enhances the response to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3312.

Abstract 6061: Novel β-Apopicropodophyllin derivative, JNC-1043, exerts anti-cancer and radiosensitizing effects

Abstract In this study, we reported novel anti-cancer drug candidate, JNC-1043{(5S)-5-(4-Benzyloxy-3,5-dimethoxy-phenyl)-5,9-dihydro-8H-furo[3',4':6,7] naphtho[2,3-d][1,3]dioxol-6-one}, synthesized from β-Apopicropodophyllin. Firstly, we analyzed anti-cancer effect of JNC-1043. We performed MTT assay to detect IC50 values on several cancer cells - breast, lung, pancreatic, blood, gastric, colorectal cancers. In these experiments, 0.1~1 μM IC50 values of JNC-1043 were calculated, and JNC-1043 treatments induced activation of caspase-3 and -8. Additionally, effective values of pharmacokinetic parameters in male rats and 2 weeks Dose Rate Finding (DRF) of JNC-1043 also detected. This result showed JNC-1043 is a promising anti-cancer candidate. Next, we tested whether radiosensitizing effect of JNC-1043. Each IC50 values of DLD-1 and HCT116 cell lines were calculated as 0.157, 0.1145, respectively. Cell counting assays were performed and then showed combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. JNC-1043 only- or IR only-treatment induced cell death by 50~60%, respectively, while combination of JNC-1043 and IR increased more 20~30% cell death than those of JNC-1043 only- or IR only-treatment in both cell lines. Clonogenic assay also performed, and then dose enhancement ratios (DER) of DLD-1 and HCT116 were calculated as 1.40, 1.53, respectively. Apoptosis also was detected with Annexin V-Propidium iodide assay. Combination of JNC-1043 and IR increased apoptosis more 30~40% than those of JNC-1043 only- or IR only-treatment. DCFDA-based assay also were performed to indicate ROS production was enhanced in combination of JNC-1043 and IR. In vivo xenograft experiment, combination of JNC-1043 and IR reduced tumor sizes less 50% and increased apoptosis more 30~40% than those of JNC-1043 only- or IR only-treatment. These results implied anti-cancer and radiosensitizing effect of JNC-1043 resulted from promotion of apoptosis via enhancement of ROS production. Citation Format: Jong Kuk Park, A-Ram Kang, Jin-Hee Kwon, Jong-Ryoo Choi, Jie-Young Song, Sang-Gu Hwang. Novel β-Apopicropodophyllin derivative, JNC-1043, exerts anti-cancer and radiosensitizing effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6061.

Evaluating the Therapeutic Potential of a Small Molecule ATR Inhibitor Combined with Radiotherapy for Head and Neck Squamous Cell Carcinoma

<h3>Purpose/Objective(s)</h3> Radiotherapy (RT) plays a major role in HNSCC treatment. Unfortunately, recurrence rates approach 50% for locally advanced, HPV-negative HNSCC, and treatment options for recurrent HNSCC are limited. New therapies are needed to enhance RT response and improve local tumor control. Ataxia telangiectasia and Rad 3-related (ATR) is a DNA damage response protein activated by replication stress (common in HNSCC) and single strand DNA breaks, such as those induced by RT. We evaluated the effect of ATR inhibitor BAY1895344 (BAY) +/- RT in preclinical HNSCC models, including the radioresistant MOC2 model. <h3>Materials/Methods</h3> HNSCC cells were treated with vehicle or serial dilutions of BAY +/- 4 Gy, and IC50 was determined by MTT assay at 72h. Sensitivity of immortalized oral keratinocyte (IMOK) cells to BAY was evaluated as a normal tissue control. To evaluate inhibition of phosphorylation of ATR downstream effector CHK1, cell lysates were harvested after treatment with BAY +/- 4 Gy (1 hr post-RT) and immunoblot performed. Radiosensitization was evaluated by clonogenic assay and quantification of yH2AX foci at 1, 3, 8, and 24 hr after 4 Gy +/- 100nM BAY. Heterotopic MOC2 tumors were induced in the hind limb of C57BL/6 mice. When tumors reached 60-110 mm³, mice were randomized to vehicle, BAY, 10 Gy, or 10 Gy + BAY (n=7-12/group). BAY was given PO (30 mg/kg BID x 3d) with RT given after the 2nd dose. Tumors were measured 3x/week, and times to tumor quintupling (TTQ) compared by ANOVA with Tukey's multiple comparisons post-test. In an ongoing experiment, mice were randomized to vehicle, BAY (50 mg/kg BID 3 days on/4 days off, continued weekly), 10 Gy, or 10 Gy+BAY (n=9/group). <h3>Results</h3> BAY inhibited CHK1 phosphorylation in a dose-dependent manner and led to persistent yH2AX foci at 24h post-RT. MTT assay showed cooperative effect between BAY and RT (IC50=63 nM for BAY vs 15nM for BAY + 4Gy) in MOC2 cells, with similar results across several primary murine HNSCC cell lines and the human FaDu cell line. IMOK cells were relatively resistant to BAY (IC50=3 uM). Clonogenic assay showed radiosensitization with 100 nM BAY (RT dose enhancement ratio = 3.7 at 4 Gy for MOC2). In the MOC2 model, BAY increased <i>in vivo</i> mean TTQ by 7.4 d vs vehicle (p=0.002), and 10 Gy + BAY increased TTQ by 7.3 d vs 10 Gy alone (p<0.001). A trend towards longer growth delay was observed 10 Gy + BAY vs BAY (mean TTQ increased by 4.3 d; p=0.08). With continued BAY dosing (3d on/4d off), median TTQ was 9.3 d with vehicle, 16.8 d with RT, and not yet reached for BAY or 10 Gy + BAY. <h3>Conclusion</h3> BAY 1895344 radiosensitizes HNSCC <i>in vitro</i> and increases tumor growth delay <i>in vivo</i> as a monotherapy or in combination with RT. Given these promising preclinical results, safety and efficacy of BAY 1895344 with stereotactic body radiation therapy (SBRT) reirradiation and pembrolizumab for recurrent HNSCC will be evaluated in an ongoing phase I CTEP trial (NCT04576091).

Role of nanoparticles in transarterial radioembolization with glass microspheres.

Transarterial Radioembolization (TARE) with 90Y-loaded glass microspheres is a locoregional treatment option for Hepatocellular Carcinoma (HCC). Post-treatment 90Y bremsstrahlung imaging using Single-Photon Emission Tomography (SPECT) is currently a gold-standard imaging modality for quantifying the delivered dose. However, the nature of bremsstrahlung photons causes difficulty for dose estimation using SPECT imaging. This work aimed to investigate the possibility of using glass microspheres loaded with 90Y and Nanoparticles (NPs) to improve the quantification of delivered doses. The Monte Carlo codes were used to simulate the post-TARE 90Y planar imaging. Planar images from bremsstrahlung photons and characteristic X-rays are acquired when 0, 1.2mol/L, 2.4mol/L, and 4.8mol/L of Gold (Au), Hafnium (Hf), and Gadolinium (Gd) NPs are incorporated into the glass microspheres. We evaluated the quality of acquired images by calculating sensitivity and Signal-to-Background Ratio (SBR). Therapeutic effects of NPs were evaluated by calculation of Dose Enhancement Ratio (DER) in tumoral and non-tumoral liver tissues. The in silico results showed that the sensitivity values of bremsstrahlung and characteristic X-ray planar images increased significantly as the NPs concentration increased in the glass microspheres. The SBR values decreased as the NPs concentration increased for the bremsstrahlung planar images. In contrast, the SBR values increased for the characteristic X-ray planar images when Hf and Gd were incorporated into the glass microspheres. The DER values decreased in the tumoral and non-tumoral liver tissues as the NPs concentration increased. The maximum dose reduction was observed at the NPs concentration of 4.8mol/L (≈7%). The incorporation of Au, Hf, and Gd NPs into the glass microspheres improved the quality and quantity of post-TARE planar images. Also, treatment efficiency was decreased significantly at NPs concentration > 4.8mol/L.

Gold-nanoparticle-enriched breast tissue in breast cancer treatment using the INTRABEAM® system: a Monte Carlo study.

Using a 50-kV INTRABEAM® system after breast-conserving surgery, breast skin injury and long treatment time remain the challenging problems when large-size spherical applicators are used. This study has aimed to address these problems using gold (Au) nanoparticles (NPs). For this, surface and isotropic doses were measured using a Gafchromic EBT3 film and a water phantom. The particle propagation code EGSnrc/Epp was used to score the corresponding doses using a geometry similar to that used in the measurements. The simulation was validated using a gamma index of 2%/2mm acceptance criterion in the gamma analysis. After validation Au-NP-enriched breast tissue was simulated to quantify any breast skin dose reduction and shortening of treatment time. It turned out that the gamma value deduced for validation of the simulation was in an acceptable range (i.e., less than one). For 20mg-Au/g-breast tissue, the calculated Dose Enhancement Ratio (DER) of the breast skin was 0.412 and 0.414 using applicators with diameters of 1.5cm and 5cm, respectively. The corresponding treatment times were shortened by 72.22% and 72.30% at 20mg-Au/g-breast tissue concentration, respectively. It is concluded that Au-NP-enriched breast tissue shows significant advantages, such as reducing the radiation dose received by the breast skin as well as shortening the treatment time. Additionally, the DERs were not significantly dependent on the size of the applicators.

DNA Dosimetry with Gold Nanoparticle Irradiated by Proton Beams: A Monte Carlo Study on Dose Enhancement

Heavy atom nanoparticles, such as gold nanoparticles, are proven effective radiosensitizers in radiotherapy to enhance the dose delivery for cancer treatment. This study investigated the effectiveness of cancer cell killing, involving gold nanoparticle in proton radiation, by changing the nanoparticle size, proton beam energy, and distance between the nanoparticle and DNA. Monte Carlo (MC) simulation (Geant4-DNA code) was used to determine the dose enhancement in terms of dose enhancement ratio (DER), when a gold nanoparticle is present with the DNA. With varying nanoparticle size (radius = 15–50 nm), distance between the gold nanoparticle and DNA (30–130 nm), as well as proton beam energy (0.5–25 MeV) based on the simulation model, our results showed that the DER value increases with a decrease of distance between the gold nanoparticle and DNA and a decrease of proton beam energy. The maximum DER (1.83) is achieved with a 25 nm-radius gold nanoparticle, irradiated by a 0.5 MeV proton beam and 30 nm away from the DNA.