Survival Fraction Research Articles

Relative Biological Effectiveness (RBE) of [64Cu]Cu and [177Lu]Lu-NOTA-panitumumab F (ab')2 radioimmunotherapeutic agents vs. γ-radiation for decreasing the clonogenic survival in vitro of human pancreatic ductal adenocarcinoma (PDAC) cells

IntroductionOur objective was to compare [64Cu]Cu-NOTA-panitumumab F(ab')2 and [177Lu]Lu-NOTA-panitumumab F(ab')2 radioimmunotherapy (RIT) agents for decreasing the clonogenic survival fraction (SF) in vitro of EGFR-positive human pancreatic ductal adenocarcinoma (PDAC) cell lines and estimate the relative biological effectiveness (RBE) vs. γ-radiation (XRT). MethodsEGFR-positive PDAC cell lines (AsPC-1, PANC-1, MIAPaCa-2, Capan-1) and EGFR-knockout PANC-1 EGFR KO cells were treated in vitro for 18 h with (0–19.65 MBq; 72 nmols/L) of [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 or XRT (0–8 Gy) followed by clonogenic assay. The SF was determined after culturing single treated cells for 14 d. Cell fractionation studies were performed for cells incubated with 1 MBq (72 nmols/L) of [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 for 1, 4, or 24 h to estimate the time-integrated activity (Ã) on the cell surface, cytoplasm, nucleus and medium. Radiation absorbed doses in the nucleus were calculated by multiplying à by S-factors calculated by Monte Carlo N Particle (MCNP) modeling using monolayer cell culture geometry. The SF of PDAC cells was plotted vs. dose and fitted to a linear quadratic model to estimate the dose required to decrease the SF to 0.1 (D10). The D10 for RIT agents were compared to XRT to estimate the RBE. DNA double-strand breaks (DSBs) caused by [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 continuous exposure for 5 h or 20 h were probed by immunofluorescence for γ-H2AX. Relative EGFR expression of PDAC cells was assessed by flow cytometry (scored + to +++) and cell doubling times for untreated cells were determined. ResultsThe D10 for [64Cu]Cu-NOTA-panitumumab F(ab′)2 ranged from 9.1 Gy (PANC-1) to 39.9 Gy (Capan-1). The D10 for [177Lu]Lu-NOTA-panitumumab F(ab′)2 ranged from 11.7 Gy (AsPC-1) to 170.8 Gy (Capan-1). The D10 for XRT ranged from 2.5 Gy (Capan-1) to 6.7 Gy (PANC-1 EGFR KO). D10 values were not correlated with EGFR expression over a relatively narrow range (++ to +++) or with cell doubling times. Based on D10 values, PANC-1 EGFR KO cells were 1.6-fold less sensitive than PANC-1 cells to [64Cu]Cu-NOTA-panitumumab F(ab′)2 and 1.9-fold less sensitive to [177Lu]Lu-NOTA-panitumumab F(ab′)2. The RBE for [64Cu]Cu-NOTA-panitumumab F(ab′)2 ranged from 0.06 for Capan-1 cells to 0.45 for PANC-1 cells. The RBE for [177Lu]Lu-NOTA-panitumumab F(ab′)2 ranged from 0.015 for Capan-1 cells to 0.28 for AsPC-1 cells. DNA DSBs were detected in PDAC cells exposed to [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 but were not correlated with the SF of the cells. ConclusionsWe conclude that at the same dose delivered to the cell nucleus [64Cu]Cu-NOTA-panitumumab F(ab′)2 and [177Lu]Lu-NOTA-panitumumab F(ab′)2 were less radiobiologically effective than XRT for decreasing the SF of human PDAC cells, but [64Cu]Cu-NOTA-panitumumab F(ab′)2 was more cytotoxic than [177Lu]Lu-NOTA-panitumumab F(ab′)2 except for AsPC-1 cells which were more sensitive to [177Lu]Lu-NOTA-panitumumab F(ab′)2. Advances in knowledge and implications for patient careThis study demonstrates that higher radiation doses may be required for RIT than XRT to achieve radiobiologically equivalent effects when used to treat PDAC.

Lethal and mutagenic effects of different LET radiations on Bacillus subtilis spores

New, useful microorganism resources have been generated by ionizing radiation breeding technology. However, the mutagenic effects of ionizing radiation on microorganisms have not been systematically clarified. For a deeper understanding and characterization of ionizing radiation-induced mutations in microorganisms, we investigated the lethal effects of seven different linear energy transfer (LET) radiations based on the survival fraction (SF) and whole-genome sequencing analysis of the mutagenic effects of a dose resulting in an SF of around 1% in Bacillus subtilis spores. Consequently, the lower LET radiations (gamma [surface LET: 0.2 keV/µm] and 4He2+ [24 keV/µm]) showed low lethality and high mutation frequency (MF), resulting in the major induction of single-base substitutions. Whereas higher LET radiations (12C5+ [156 keV/µm] and 12C6+ [179 keV/µm]) showed high lethality and low MF, resulting in the preferential induction of deletion mutations. In addition, 12C6+ (111) ion beams likely possess characteristics of both low- and high-LET radiations simultaneously. A decrease in the relative biological effectiveness and an evaluation of the inactivation cross section indicated that 20Ne8+ (468 keV/µm) and 40Ar13+ (2214 keV/µm) ion beams had overkill effects. In conclusion, in the mutation breeding of microorganisms, it should be possible to regulate the proportions, types, and frequencies of induced mutations by selecting an ionizing radiation of an appropriate LET in accordance with the intended purpose.

Dependence of Induced Biological Damage on the Energy Distribution and Intensity of Clinical Intra-Operative Radiotherapy Electron Beams.

The survival fraction of epithelial HaCaT cells was analysed to assess the biological damage caused by intraoperative radiotherapy electron beams with varying energy spectra and intensities. These conditions were achieved by irradiating the cells at different depths in water using nominal 6 MeV electron beams while consistently delivering a dose of 5 Gy to the cell layer. Furthermore, a Monte Carlo simulation of the entire irradiation procedure was performed to evaluate the molecular damage in terms of molecular dissociations induced by the radiation. A significant agreement was found between the molecular damage predicted by the simulation and the damage derived from the analysis of the survival fraction. In both cases, a linear relationship was evident, indicating a clear tendency for increased damage as the averaged incident electron energy and intensity decreased for a constant absorbed dose, lowering the dose rate. This trend suggests that the radiation may have a more pronounced impact on surrounding healthy tissues than initially anticipated. However, it is crucial to conduct additional experiments with different target geometries to confirm this tendency and quantify the extent of this effect.

Triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro.

Combined chemoradiation offers a promising therapeutic strategy for dogs with glioma. The alkylating agents temozolomide (TMZ) and lomustine (CCNU) penetrate the blood-brain barrier, and doses for dogs are established. Whether such combinations are clinically advantageous remains to be explored together with tumour-specific markers. To investigate if triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro. We evaluated the sensitising effect of CCNU alone and in combination with TMZ-irradiation in canine glioma J3T-BG cells and long-term drug-exposed subclones by using clonogenic survival and proliferation assays. Bisulphite-SEQ and Western Blot were used to investigate molecular alterations. TMZ (200μM) or CCNU alone (5μM) reduced the irradiated survival fraction (4Gy) from 60% to 38% (p= 0.0074) and 26% (p= 0.0002), respectively. The double-drug combination reduced the irradiated survival fraction (4Gy) more potently to 12% (p< 0.0001). After long-term drug exposure, both subclones show higher IC50 values against CCNU and TMZ. For CCNU-resistant cells, both, single-drug CCNU (p= 0.0006) and TMZ (p= 0.0326) treatment combined with irradiation (4Gy) remained effective. The double-drug-irradiation combination reduced the cell survival by 86% (p< 0.0001), compared to 92% in the parental (nonresistant) cell line. For TMZ-resistant cells, only the double-drug combination with irradiation (4Gy) reduced the cell survival by 88% (p= 0.0057) while single-drug treatment lost efficacy. Chemoresistant cell lines demonstrated higher P-gp expression while MGMT-methylation profile analysis showed a general high methylation level in the parental and long-term treated cell lines. Our findings indicate that combining CCNU with TMZ-irradiation significantly reduces canine glioma cell survival. Such a combination could overcome current challenges of therapeutic resistance to improve overall patient survival.

A spatiotemporal computational model of focused ultrasound heat-induced nano-sized drug delivery system in solid tumors

Focused Ultrasound (FUS)-triggered nano-sized drug delivery, as a smart stimuli-responsive system for treating solid tumors, is computationally investigated to enhance localized delivery of drug and treatment efficacy. Integration of thermosensitive liposome (TSL), as a doxorubicin (DOX)-loaded nanocarrier, and FUS, provides a promising drug delivery system. A fully coupled partial differential system of equations, including the Helmholtz equation for FUS propagation, bio-heat transfer, interstitial fluid flow, drug transport in tissue and cellular spaces, and a pharmacodynamic model is first presented for this treatment approach. Equations are then solved by finite element methods to calculate intracellular drug concentration and treatment efficacy. The main objective of this study is to present a multi-physics and multi-scale model to simulate drug release, transport, and delivery to solid tumors, followed by an analysis of how FUS exposure time and drug release rate affect these processes. Our findings not only show the capability of model to replicate this therapeutic approach, but also confirm the benefits of this treatment with an improvement of drug aggregation in tumor and reduction of drug delivery in healthy tissue. For instance, the survival fraction of tumor cells after this treatment dropped to 62.4%, because of a large amount of delivered drugs to cancer cells. Next, a combination of three release rates (ultrafast, fast, and slow) and FUS exposure times (10, 30, and 60 min) was examined. Area under curve (AUC) results show that the combination of 30 min FUS exposure and rapid drug release leads to a practical and effective therapeutic response.

Relative biological effectiveness for epithermal neutron beam contaminated with fast neutrons in the linear accelerator-based boron neutron capture therapy system coupled to a solid-state lithium target.

This study aimed to quantify the relative biological effectiveness (RBE) for epithermal neutron beam contaminated with fast neutrons in the accelerator-based boron neutron capture therapy (BNCT) system coupled to a solid-state lithium target. The experiments were performed in National Cancer Center Hospital (NCCH), Tokyo, Japan. Neutron irradiation with the system provided by Cancer Intelligence Care Systems (CICS), Inc. was performed. X-ray irradiation, which was assigned as the reference group, was also performed using a medical linear accelerator (LINAC) equipped in NCCH. The four cell lines (SAS, SCCVII, U87-MG and NB1RGB) were utilized to quantify RBE value for the neutron beam. Before both of those irradiations, all cells were collected and dispensed into vials. The doses of 10% cell surviving fraction (SF) (D10) were calculated by LQ model fitting. All cell experiments were conducted in triplicate at least. Because the system provides not only neutrons, but gamma-rays, the contribution from the gamma-rays to the survival fraction were subtracted in this study. D10 value of SAS, SCCVII, U87-MG and NB1RGB for the neutron beam was 4.26, 4.08, 5.81 and 2.72Gy, respectively, while that acquired by the X-ray irradiation was 6.34, 7.21, 7.12 and 5.49Gy, respectively. Comparison of both of the D10 values, RBE value of SAS, SCCVII, U87-MG and NB1RGB for the neutron beam was calculated as 1.7, 2.2, 1.3 and 2.5, respectively, and the average RBE value was 1.9. This study investigated RBE of the epithermal neutron beam contaminated with fast neutrons in the accelerator-based BNCT system coupled to a solid-state lithium target.

Differentiating bladder cancer (BLCA) tumor immune microenvironment (TME) profiles based on predicted intrinsic radiosensitivity (RS).

e16622 Background: Radiation is an essential part of organ preservation therapy for muscle-invasive BLCA patients. There is increasing interest in use of immunotherapy in this setting and defining biomarkers of RS and immunity will be crucial to guide patient selection. We investigated whether predicted RS of BLCA patients can differentiate TME profiles based on RNA-seq. Methods: Twenty genomically-verified BLCA cell lines were profiled with log(TPM+1) normalized RNA-seq counts. Colony forming assays (CFA) for each cell line were done in triplicate with RS indices measured via area-under-the-cell-survival curve (AUC) and surviving fraction (SF) at varying doses (0-8Gy). The RS signature (RSS) was derived in two steps. First, NMF-decomposition of the RNA-seq data was done via CoGAPS v.3.6 run at k = 13 gene sets for 10,000 iterations then gene sets significantly correlated (p ≤ 0.05) with RS indices defined as an absolute Spearman’s rho &gt;0.3 in at least 4/5 indices between AUC, SF2, SF4, SF6, and SF8 were combined. Secondly, the top 100-genes overexpressed by top-third most sensitive cell lines were identified by ComparativeMarkerSelection. The RSS contained genes shared between approaches and the RS-scores for the 408-patient TCGA-BLCA cohort were defined by averaging z-score normalized gene expression in the RSS. Patients were grouped as RS-high (top-quartile RS scores) and -low (bottom-quartile RS scores. Enrichment of the TME was assessed via EcoTyper algorithm and significant differences were defined a priori as: 1) Student’s t-test p ≤ 0.1 with Benjamini-Hochberg correction with an FDR of 0.2; 2) absolute correlation of r ≥ 0.4 between cell-state and RS-score; 3) ≥30% difference in enrichment between RS-high and RS-low. Results: Satisfactory CFA were obtained for 18 cell lines (range of RS indices: AUC = 3.40-5.94; SF2 = 0.32-0.75; SF4 = 0.09-0.42; SF6 = 0.02-0.19; SF8 = 0.002-0.09). CoGAPS analysis revealed 4 gene sets that were significantly correlated with RS totaling 1,997 genes. The RSS ultimately contained 78 genes and TCGA-BLCA RS scores ranged from -0.3 to 5.2. RS-high patients had significant enrichment for polymorphonuclear neutrophils (PMNs S02 state; 46-fold) and mast cells (S01 state; 77-fold). RS-low patients had significant enrichment for multiple cell-states including exhausted CD4 T-cells (S01 state; 1.31-fold; associated with CXCR6 &amp; CTLA-4) and cancer-associated fibroblasts (S03 state, 1.33-fold; associated with POSTN &amp; COL10A1). Conclusions: We demonstrate that predicted RS of BLCA patients is associated with a differential TME profile characterized by PMN inflammation and CD4 T-cell mediated immunosuppression among other cellular states. This potential link between RS and immunity may have broader implications for biomarker-guided immunotherapy as part of organ-preservation for BLCA patients although further validation is needed.

LIFE TABLE ATTRIBUTES AND FEEDING EFFICIENCY OF OENOPIA SAUZETI (MULSANT) (COLEOPTERA: COCCINELLIDAE) FED ON APHID (APHIS GOSSYPII)

Ladybird beetles are play vital role in agroecosystem due to successful and efficient feeding behavior against aphids, the present research experiment was carried to find out the life history and feeding efficacy of oenopiasauzetifed on Aphis gossypii,at the laboratory of Biological Control, integrated pest management programme (IPMP), National Agriculture Research Centre (NARC), Islamabad. The results of the experiment revealed that the mean developmental time of immature stage 3rd and 4th instar have a significant difference with 3.72±0.11 and 3.87±0.036 days, while the egg to adult emergence was 39±0.35 days. The longevity has significant difference in male and female with the higher values at female 34.0 days. Similarly, the feeding efficiency was maximum in O. sauzeti females which feeds on 2227.1 mean number of Aphis gossypii, while the (409.25±1.42) mean numbers of aphids were consumed by larva. The maximum aphid consumption was observed at 4th instar larva (198.98±0.51 aphid) followed by 3rd instar larva (128.57±0.59 aphid). The mean oviposition time of O. sauzetion A. gossypii was recorded as 21.36 days and average eggs laid female-1 day-1 was 20.53, while the total laid eggs given by a female were 253.60. The apparent mortality (100qx) was maximum at egg stage (24) and the mortality survival ratio (MSR) was highest at pupal stage (0.53), similarly, the indispensable mortality (IM) was also observed high at pupal stage. The maximum survival fraction (Sx) was observed at the 4th larval stage (0.91) and the maximum life expectancy (ex) was observed at 1st larval instar (4.51) days. The total killer power (k-value) of the O. sauzetifor the complete generation was 0.49. This will help to develop an ecofriendly managing approach for the minimizing the aphids infestationin economical important crops.

PiRNA-14633 underexpression as an independent prognostic marker in non-small cell lung cancer.

e21223 Background: To determine the expression and function of piRNA-14633 in non-small cell lung cancer (NSCLC) samples were taken from cancer lesions (n = 30) and adjacent normal tissue (n = 30) in NSCLC patients. Methods: piRNA-14633 expression was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The effect of piRNA-14633 overexpression was examined in vitro utilizing a human NSCLC cell line H1650 stably transfected with a recombinant lentivirus and compared to empty vector-transfected controls. piRNA-14633 overexpression was verified by RT-PCR. The expression of proteins involved in cell cycle regulation (CDK4) and apoptosis (Bcl-6) and cell migration (MMP-9) in cells was analyzed by Western blot. The effect of piRNA-14633 overexpression on cell viability and proliferation was assessed by an MTT assay cells. Flow cytometric analyses were used to evaluate the effect of piRNA-14633 expression on cell cycle distribution and apoptosis. The migration and invasion potential of piRNA-14633 cells was examined by plating cells in Matrigel-coated chambers. Results: The level of piRNA-14633 expression was found to be significantly lower in NSCLC tissue than normal tissues (P &lt; 0.05). Decreased expression of piRNA-14633 was significantly correlated with lymph node metastasis, clinical stage, and histological grade of patients with NSCLC (P &lt; 0.05). Meanwhile, loss of piRNA-14633 expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P &lt; 0.05). The result of biological function shown that H1650 cell-transfected piRNA-14633 had a lower survival fraction, higher percentage of the G0/G1 phases, higher cell apoptosis, significant decrease in migration and invasion, and lower CDK4, Bcl-6, and MMP-9 expression compared with H1650 cell without piRNA-14633 (P &lt; 0.05). Conclusions: Reduced piRNA-14633 expression is associated with increased disease severity, suggesting it is a negative regulator of NSCLC and can serve as a prognostic indicator.

Association of PIWI-interacting RNA-23210 with tumor metastasis in esophageal squamous cell carcinoma.

e16060 Background: his study aimed to analyze the expression, clinical significance of PIWI-interacting RNA-23210 (piRNA-23210) in esophageal squamous cell carcinoma (ESCC) and the biological effect in its cell line by piRNA-23210 overexpression. Methods: Quantitative real-time RT-PCR (qRT-PCR) was used to analyze piRNA-23210 expression in 60 cases of ESCC and 60 cases of normal tissues to study the relationship between piRNA-23210 expression and clinical factors. Recombinant lentiviral vector was constructed to overexpress piRNA-23210 and then infect ESCC TE-1 cell line. qRT-PCR was used to detect the level of piRNA-23210. 3-[4,5-dimethylthiazol -2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis, cell cycle, and migration and invasion assays were also conducted as to the influence of the upregulated expression of piRNA-23210 that might be found on TE-1 cells biological effect. Results: The level of piRNA-23210 expression was found to be significantly lower in ESCC tissue than normal tissues (P &lt; 0.05). Decreased expression of piRNA-23210 was significantly correlated with lymph node metastasis, clinic stage, and histological grade of patients with ESCC (P &lt; 0.05). Meanwhile, loss of piRNA-23210 expression correlated significantly with poor overall survival time by Kaplan–Meier analysis (P &lt; 0.05). The result of biological function show that TE-1 cell transfected piRNA-23210 had a lower survival fraction; higher percentage of the G0/G1 phases; higher cell apoptosis; significant decrease in migration and invasion; and lower cyclin D1, Bcl-2, and MMP-7 protein expression compared with TE-1 cell untransfected piRNA-23210 (P &lt; 0.05). Conclusions: piRNA-23210 expression decreased in ESCC and correlated significantly with lymph node metastasis, clinical stage, poor overall survival, cell proliferation, cell cycles, cell apoptosis and migration and invasion in ESCC cell by regulating cyclinD1, Bcl-2, and MMP-7 expression, suggesting that piRNA-23210 may play important roles as a negative regulator to ESCC cell.

7-Geranyloxcycoumarin enhances radio sensitivity in human prostate cancer cells.

Prostate cancer is the second most prevalent and the fifth deadliest cancer among men worldwide. To improve radiotherapy outcome, we investigated the effects of 7-geranyloxycoumarin, also known as auraptene (AUR), on radiation response of prostate cancer cells. PC3 cells were pretreated with 20 and 40 µM AUR for 24, 48 and 72h, followed by X-ray exposure (2, 4 and 6Gy). After 72h recovery, cell viability was determined by alamar Blue assay. Flow cytometric analysis was performed to assess apoptosis induction, clonogenic assay was carried out to investigate clonogenic survival, and the expression of P53, BAX, BCL2, CCND1 and GATA6 was analyzed by quantitative polymerase chain reaction (qPCR). Cell viability assay indicated that toxic effects of radiation was enhanced by AUR, which was also confirmed by increased numbers of apoptotic cells and reduced amount of survival fraction. The qPCR results demonstrated significant induction of P53 and BAX, while the expression of BCL2, GATA6, and CCND1 was significantly downregulated. The findings of the present study indicated, for the first time, that AUR improved radio sensitivity in prostate cancer cells, and thus, has the potential to be used in future clinical trials.

Comparison of LEMI, LEMII and LEMIII to prediction of cell survival fraction of HTB140 cell line with GEANT4 and SURVIVAL code in proton therapy

The aim of this paper is to present the comparison of three models of Local Effect Model (LEMI, LEMII, LEMIII) with penetration depth of proton in a typical Spread-Out Bragg Peak (SOBP) for cell Survival Fraction (CSF) of HTB140 cell line.To evaluate the CSF, two tools, GEANT4 code and SURVIVAL code, were used. The dose and Linear Energy Transfer (LET) output of GEANT4 were used as input of SURVIVAL code for calculation of CSF parameters (alpha and beta) using three versions of LEM (LEMI, LEMII, and LEMIII). By using the Linear-Quadratic (LQ) model and output of survival code, the curve of CSF was predicted for HTB140 cell line and reasonable agreement with experimental data of CATANA (Centro di AdroTerapia ed Applicazioni Nucleari Avanzate) center was achieved. At 2 Gy, by moving from proximal to distal end of SOBP, the CSF of LEMI decreased from 0.94 to 0.92 and then increased to 0.97 at distal declining edge but LEMII and LEM III increased from 0.89 to 0.96 and 0.9 to 0.96 (proximal and distal declining edge) respectively.This is the first study to compare LEMI, LEMII, and LEMIII models for CSF curve of proton with penetration depth and was coupled both GEANT4 and SURVIVAL code to predict the CSF parameter. According to results, the prediction of the LEMII and LEMIII were in better agreement with experimental results as compared to the LEMI model.

Reproducibility study of Monte Carlo simulations for nanoparticle dose enhancement and biological modeling of cell survival curves

Nanoparticle-derived radiosensitization has been investigated by several groups using Monte Carlo simulations and biological modeling. In this work we replicated the physical simulation and biological modeling of previously published research for 50 nm gold nanoparticles irradiated with monoenergetic photons, various 250 kVp photon spectra, and spread-out Bragg peak (SOBP) protons. Monte Carlo simulations were performed using TOPAS and used condensed history Penelope low energy physics models for macroscopic dose deposition and interaction with the nanoparticle; simulation of the microscopic dose deposition from nanoparticle secondaries was performed using Geant4-DNA track structure physics. Biological modeling of survival fractions was performed using a local effect model-type approach for MDA-MB-231 breast cancer cells. Physical simulation results agreed extraordinarily well at all distances (1 nm to 10 μm from nanoparticle) for monoenergetic photons and SOBP protons in terms of dose per interaction, dose kernel ratio (often labeled dose enhancement factor), and secondary electron spectra. For 250 kVp photons the influence of the gold K-edge was investigated and found to appreciably affect the results. Calculated survival fractions similarly agreed well within one order of magnitude at macroscopic doses (i.e. without nanoparticle contribution) from 1 Gy to 10 Gy. Several 250 kVp spectra were tested to find one yielding closest agreement with previous results. This highlights the importance of a detailed description of the low energy (< 150 keV) component of photon spectra used for in-silico, as well as in-vitro, and in-vivo studies to ensure reproducibility of the experiments by the scientific community. Both, Monte Carlo simulations of physical interactions of the nanoparticle with photons and protons, as well as the biological modelling of cell survival curves agreed extraordinarily well with previously published data. Further investigation of the stochastic nature of nanoparticle radiosenstiziation is ongoing.

O097 A drug screen to identify novel radiosensitisers in colorectal cancer cells

Abstract Introduction Locally advanced rectal cancer is typically treated with neoadjuvant chemoradiotherapy, where chemotherapy acts synergistically to radiosensitise cancer cells. Response to chemoradiotherapy is variable, with 8-20% of cases having a complete pathological response, but 20-30% demonstrate no response or progression. Novel radiosensitisers can potentially improve response, tolerability of radiotherapy and de-escalate treatments, including the need for major surgery. This study aims to perform a screen of available FDA-approved drugs to identify novel radiosensitisers in rectal cancer. Methods A drug screen of forty FDA-approved drugs was performed. HCT-116 cells were cultured and treated as a monolayer with 0.3 μM and 1.0 μM of a specific drug for 16 hours. Clonogenic assays were performed in 24 well plates after photon irradiation at 0 and 1 Gy. Relative colony formation (survival fraction) was employed to measure drug efficacy and radiosensitisation. Following the screen, two drugs were selected for dose optimisation and further mechanistic evaluation with DNA repair kinetics and DNA damage using immunofluorescence of γH2Ax foci. Results Abexinostat and fomepizole were selected from the screen for dose optimisation based on their radiosensitising capabilities. Dose optimisations determined that both drugs had an optimal concentration of 1.0 μM and required an optimum of 6 hours of treatment and showed significant radiosensitisation. There were significantly more γH2Ax foci after 24 hours in cells treated with Abexinostat than control, demonstrating persistent DNA damage. Conclusion Abexinostat and fomepizole show promise as radiosensitizers. Further experimentation is ongoing to determine the mechanisms for these drugs and translational benefit.

Radiosensitivity Enhancement using Triptorelin Conjugated Bismuth Sulfide Nanoparticles (Bi2S3@BSA) in Radiotherapy for Breast Cancer Cells

The aim of this study was to assess the radiosensitivity of bismuth sulfide nanoparticles conjugated with a synthetic agonist analog of gonadotropin-releasing hormones in targeted radiotherapy for breast cancer. After synthesis and characterization of nanoparticles, cytotoxicity of nanoparticles was measured by MTT assay, and the survival fraction was determined by colony formation assay. Finally, flow cytometry was performed to identify the mechanism of radiosensitization. Characterization results determined the spherical shape of Bi2S3@BSA with an average size of 8.649 ± 1.6 nm, and Fourier transform infrared confirmed the successful binding of triptorelin to the surface of the nanoparticles. MTT test results show that the Bi2S3@BSA–triptorelin did not cause any toxicity ( P &lt; 0.05 ) even up to 75 μg/ml. At all doses of ionizing radiation, colony formation assays showed that the nontoxic concentration of Bi2S3@BSA–triptorelin significantly increased cell death in MCF-7 cells compared to Bi2S3@BSA ( P &lt; 0.05 ). The apoptosis test also confirmed colony formation assay results at all doses and introduced apoptosis as a mechanism of radiosensitivity produced by nanoparticles. Certainly, targeted bismuth sulfide nanoparticles can be a good candidate for increasing radiosensitivity against tumor cells.

High-frequency irreversible electroporation improves survival and immune cell infiltration in rodents with malignant gliomas.

Irreversible electroporation (IRE) has been previously investigated in preclinical trials as a treatment for intracranial malignancies. Here, we investigate next generation high-frequency irreversible electroporation (H-FIRE), as both a monotherapy and a combinatorial therapy, for the treatment of malignant gliomas. Hydrogel tissue scaffolds and numerical modeling were used to inform in-vivo H-FIRE pulsing parameters for our orthotopic tumor-bearing glioma model. Fischer rats were separated into five treatment cohorts including high-dose H-FIRE (1750V/cm), low-dose H-FIRE (600V/cm), combinatorial high-dose H-FIRE + liposomal doxorubicin, low-dose H-FIRE + liposomal doxorubicin, and standalone liposomal doxorubicin groups. Cohorts were compared against a standalone tumor-bearing sham group which received no therapeutic intervention. To further enhance the translational value of our work, we characterize the local and systemic immune responses to intracranial H-FIRE at the study timepoint. The median survival for each cohort are as follows: 31 days (high-dose H-FIRE), 38 days (low-dose H-FIRE), 37.5 days (high-dose H-FIRE + liposomal doxorubicin), 27 days (low-dose H-FIRE + liposomal doxorubicin), 20 days (liposomal doxorubicin), and 26 days (sham). A statistically greater overall survival fraction was noted in the high-dose H-FIRE + liposomal doxorubicin (50%, p = 0.044), high-dose H-FIRE (28.6%, p = 0.034), and the low-dose H-FIRE (20%, p = 0.0214) compared to the sham control (0%). Compared to sham controls, brain sections of rats treated with H-FIRE demonstrated significant increases in IHC scores for CD3+ T-cells (p = 0.0014), CD79a+ B-cells (p = 0.01), IBA-1+ dendritic cells/microglia (p = 0.04), CD8+ cytotoxic T-cells (p = 0.0004), and CD86+ M1 macrophages (p = 0.01). H-FIRE may be used as both a monotherapy and a combinatorial therapy to improve survival in the treatment of malignant gliomas while also promoting the presence of infiltrative immune cells.

Modelling oxygen effects on the in- and out-of-field radiosensitivity of cells exposed to intensity-modulated radiation fields

Objective. The delivery of intensity-modulated radiation fields has improved the conformity of dose to tumour targets during radiotherapy (RT). Previously, it has been shown that intercellular communication between cells positioned in- and outside of the radiation field impacts cellular radiosensitivity under hypoxic and normoxic conditions. However, the mechanism of intercellular communication in hypoxia remains to be fully understood. In this study, the cell-killing effects of intercellular communication in hypoxia were modelled in an effort to better understand the underlying mechanisms of response. Approach. By irradiating a 50% area of the culture dish (half-field exposure), experimental dose-response curves for cell survival and residual DNA double-strand breaks (DSBs) were generated in prostate (DU145) and non-small cell lung cancer (H1299) cells. The oxygen enhancement ratio (OER) was determined from early DSB yields (corresponding to relative direct damage) and used to model the in- and out-of-field radiosensitivity. Main results. The developed integrated microdosimetric-kinetic (IMK) model successfully predicted the experimental dose responses for survival and lethal lesions, and provides a mechanistic interpretation that the probability of hits for releasing cell-killing signals is dependent on oxygen. This experimental and modelling study also suggests that residual DSBs correspond to logarithmic survival fraction (meaning lethal lesions) for in- and out-of-field cells. Our data suggest that the OER value determined using uniform-field exposure can be applied to predict the in- and out-of-field radiosensitivity of cells following exposure to intensity modulated beams. Significance. The developed IMK model facilitates a more precise understanding of intercellular signalling following exposure to intensity-modulated radiation fields.

An artificial intelligence-based model for cell killing prediction: development, validation and explainability analysis of the ANAKIN model

The present work develops ANAKIN: an Artificial iNtelligence bAsed model for (radiation-induced) cell KIlliNg prediction. ANAKIN is trained and tested over 513 cell survival experiments with different types of radiation contained in the publicly available PIDE database. We show how ANAKIN accurately predicts several relevant biological endpoints over a wide broad range on ion beams and for a high number of cell-lines. We compare the prediction of ANAKIN to the only two radiobiological models for Relative Biological Effectiveness prediction used in clinics, that is the Microdosimetric Kinetic Model and the Local Effect Model (LEM version III), showing how ANAKIN has higher accuracy over the all considered cell survival fractions. At last, via modern techniques of Explainable Artificial Intelligence (XAI), we show how ANAKIN predictions can be understood and explained, highlighting how ANAKIN is in fact able to reproduce relevant well-known biological patterns, such as the overkilling effect.

Carborane-Containing Hydroxamate MMP Ligands for the Treatment of Tumors Using Boron Neutron Capture Therapy (BNCT): Efficacy without Tumor Cell Entry.

New carborane-bearing hydroxamate matrix metalloproteinase (MMP) ligands have been synthesized for boron neutron capture therapy (BNCT) with nanomolar potency against MMP-2, -9 and -13. New analogs are based on MMP inhibitor CGS-23023A, and two previously reported MMP ligands 1 (B1) and 2 (B2) were studied in vitro for BNCT activity. The boronated MMP ligands 1 and 2 showed high in vitro tumoricidal effects in an in vitro BNCT assay, exhibiting IC50 values for 1 and 2 of 2.04 × 10-2 mg/mL and 2.67 × 10-2 mg/mL, respectively. The relative killing effect of 1 to L-boronophenylalanine (BPA) is 0.82/0.27 = 3.0, and that of 2 is 0.82/0.32 = 2.6, whereas the relative killing effect of 4 is comparable to boronophenylalanine (BPA). The survival fraction of 1 and 2 in a pre-incubation boron concentration at 0.143 ppm 10B and 0.101 ppm 10B, respectively, were similar, and these results suggest that 1 and 2 are actively accumulated through attachment to the Squamous cell carcinoma (SCC)VII cells. Compounds 1 and 2 very effectively killed glioma U87 delta EGFR cells after BNCT. This study is noteworthy in demonstrating BNCT efficacy through binding to MMP enzymes overexpressed at the surface of the tumor cell without tumor cell penetration.

Abstract 2830: Fluoxetine mediate radiosensitivity and inhibit metastasis of osteosarcoma cells via RANK/RANKL signaling

Abstract Osteosarcoma (OS) is most common primary malignant bone tumour OS was flagrantly byaffecting pediatric and young adolescents patients with its outrageous metastatic rate and poorprognosis. It was aggressive in local invasion and further metastasis, especially pulmonarymetastasis. Radiotherapy (RT) was a clinical treatment method that using high-energy ionizingradiation to control tumour growth with low risk and considerably successful rate.Contradictorily, OS was recognized as radio-resistant tumour. Meanwhile, majority of OScases were diagnosed suffering depression and influence patient’s conditions critically. Hence,aim of this prospective study was to identify that combinational therapeutic efficacy ofradiation therapy and fluoxetine (Prozac) through mediating radiosensitivity and inhibitmetastasis of OS cells via RANK/RANKL signaling. OS cell lines U2OS and MG63 were usedin this study. To investigate the cells survival fraction, clonogenic assay were performed. Next,to examine metastasis and invasions of cells wound healing assay and transwell assay werecarried out. Furthermore, TUNEL assay was used to evaluate DNA damage of OS cells.Fluoxetine effectively inhibited the metastasis and invasion ability of OS cells. Thecombinational therapy of fluoxetine and RT successfully decreased the surviving fraction ofOS cells and induced the damage of DNA. Additionally, the protein expression ofRANK/RANKL was markedly suppressed by fluoxetine as illustrated by Western blottingassay. In sum, the combination treatment of radiotherapy with the interrelation of fluoxetinewas showing its potential and consider as new treatment strategy might elevate treatmentefficacy for OS. Keywords: Osteosarcoma (OS), Radiotherapy (RT), Fluoxetine, RANK/RANKL Citation Format: Peggy Tan, Jai-Jen Tsai, Fei-Ting Hsu, Yu-Chiang Liu. Fluoxetine mediate radiosensitivity and inhibit metastasis of osteosarcoma cells via RANK/RANKL signaling [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 2830.