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Purpose Triple negative breast cancers (TNBC) are more often found among BRCA gene mutation carriers; even among non-mutation carriers, TNBCs are thought to carry a phenotype of BRCAness. Inhibitors of Poly(adenosine diphosphate-ribose) polymerase (PARPi) have improved survival outcomes for both advanced and early stage breast cancer associated with a BRCA mutation, as well as BRCA mutated ovarian and prostate cancers. Radiotherapy (RT) is a widely used treatment to locally control cancers, but it is unclear how different types of RT, including clinical x-rays and protons, interact with PARPi and how these affect antitumor immunity. Protons induce more clustered DNA lesions, including clustered double and single strand breaks (DSBs and SSBs) and clustered base damages, than x-rays due to their higher ionization density. If not properly repaired, DSBs can generate micronuclei (MN), which activate the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon (IFN) genes (STING) (cGAS-STING) pathway, leading to antitumor immunity. Residual DNA damage also induces cellular senescence, which is a known tumor suppressor mechanism. In this study we investigated the effects of combining a PARPi with x-rays or protons on clonogenic cell survival, residual DNA damage, senescence, cGAS co-localization with MN and delay in tumor growth in the context of BRCA1 mutation. Methods In vitro, BRCA1 deficient (HCC1937 and MDA-MB-436) and proficient (HCC1937-BRCA and MDA-MB-436-BRCA) breast cancer cell lines were treated with a PARPi (Olaparib, 0.1 - 5.0 μM) and irradiated with 6 MV x-rays or protons (9.9 keV/um) with doses from 0.5 to 5 Gy. HCC1937-BRCA was gifted from Dr. Stecklein (The University of Kansas Cancer Center) and MDA-MB-436-BRCA was gifted from Dr. Johnson (Fox Chase Cancer Center). We then quantified clonogenic cell survival, gH2AX and 53BP1 foci at 24 h after irradiation, MN number and MN-cGAS co-localization (cGAS+ MN) at 24 and 74 h after irradiation, and RT-induced senescence. In vivo, we used a preclinical TNBC mouse model (4T1 tumors on the leg of BALB/c mice) to assess survival and tumor growth delay on animals treated with DMSO, PARPi alone (Olaparib, 100 mg/kg), DMSO+x-rays, and PARPi+x-rays. Experiments with protons are ongoing. PARPi was administrated via oral gavage 2 h prior to irradiation (1 × 11 Gy). Results Survival fraction was lower after protons compared to x-rays (RBED10%>1) in HCC1937-BRCA, HCC1937 and MDA-MB-436 cells. Regardless of the radiation type, survival fraction was lower for HCC1937 compared to HCC1937-BRCA cells. PARPi treatment appears to result in greater radiosensitization of cells exposed to protons than those exposed to x-rays as assessed by clonogenic cell survival. The number of MN per nucleus was higher in HCC1937 than HCC1937-BRCA at 24 and 72 h after protons and x-rays at both 2 and 5 Gy, and this effect that was amplified with PARPi vs. without PARPi. Moreover, the number of cGAS+ MN was greater after protons than x-rays for HCC1937 but not HCC1937-BRCA at 24 and 72 h. The proportion of senescent cells for HCC1937-BRCA was higher (1.4 fold) after PARPi+protons compared to PARPi+x-rays. In vivo, x-rays lead to a delay in tumor growth, which was higher for the PARPi+x-rays group compared to PARPi alone or DMSO+x-rays groups. Conclusion In vitro, a PARPi treatment in BRCA1 mutated cell lines leads to a greater sensitivity to radiation compared to their counterparts with recovered BRCA1 function. This sensitivity is higher after protons compared to x-rays (survival fraction and number of micronuclei). In vivo, PARPi treatment combined with x-rays lead to higher delay in tumor growth compared to x-rays alone. These preliminary data are promising results on the effect of radiation combined with PARPi on BRCA1 mutated models. Citation Format: Mariam Ben Kacem, Scott J. Bright, Broderick X Turner, David B Flint, Mandira Manandhar, David Martinus, Gabriel O Sawakuchi, Simona F Shaitelman. Parp inhibition sensitizes brca deficient cancer cell lines and tumors to clinical x-ray and proton irradiation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-01.

Purpose To show that intrinsic radiosensitivity varies greatly for protons and carbon (C) ions in addition to photons, and that DNA repair capacity remains important in governing this variability. Methods We measured or obtained from the literature clonogenic survival data for a number of human cancer cell lines exposed to photons, protons (9.9 keV/μm), and C-ions (13.3-77.1 keV/μm). We characterized their intrinsic radiosensitivity by the dose for 10% or 50% survival (D10% or D50% ), and quantified the variability at each radiation quality by the coefficient of variation (COV) in D10% and D50% . We also treated cells with DNA repair inhibitors prior to irradiation to assess how DNA repair capacity affects their variability. Results We found no statistically significant differences in the COVs of D10% or D50% between any of the radiation qualities investigated. The same was true regardless of whether the cells were treated with DNA repair inhibitors, or whether they were stratified into histologic subsets. Even within histologic subsets, we found remarkable differences in radiosensitivity for high LET C-ions that were often greater than the variations in RBE, with brain cancer cells varying in D10% (D50% ) up to 100% (131%) for 77.1 keV/μm C-ions, and non-small cell lung cancer and pancreatic cancer cell lines varying up to 55% (76%) and 51% (78%), respectively, for 60.5 keV/μm C-ions. The cell lines with modulated DNA repair capacity had greater variability in intrinsic radiosensitivity across all radiation qualities. Conclusions Even for cell lines of the same histologic type, there are remarkable variations in intrinsic radiosensitivity, and these variations do not differ significantly between photon, proton or C-ion radiation. The importance of DNA repair capacity in governing the variability in intrinsic radiosensitivity is not significantly diminished for higher LET radiation.

Proton relative biological effectiveness (RBE) is known to depend on physical factors of the proton beam, such as its linear energy transfer (LET), as well as on cell-line specific biological factors, such as their ability to repair DNA damage. However, in a clinical setting, proton RBE is still considered to have a fixed value of 1.1 despite the existence of several empirical models that can predict proton RBE based on how a cell's survival curve (linear-quadratic model [LQM]) parameters α and β vary with the LET of the proton beam. Part of the hesitation to incorporate variable RBE models in the clinic is due to the great noise in the biological datasets on which these models are trained, often making it unclear which model, if any, provides sufficiently accurate RBE predictions to warrant a departure from RBE = 1.1.Here, we introduce a novel model of proton RBE based on how a cell's intrinsic radiosensitivity varies with LET, rather than its LQM parameters.We performed clonogenic cell survival assays for eight cell lines exposed to 6 MV x-rays and 1.2, 2.6, or 9.9 keV/µm protons, and combined our measurements with published survival data (n = 397 total cell line/LET combinations). We characterized how radiosensitivity metrics of the form DOur analyses of the trends in the data show that DWe established a new empirical proton RBE model that is more accurate than previous empirical models, and that predicts much higher RBE values in the distal edge of clinical proton beams.

We assessed whether adding sodium borocaptate (BSH) or 4-borono-l-phenylalanine (BPA) to cells irradiated with proton beams influenced the biological effectiveness of those beams against prostate cancer cells to investigate if the alpha particles generated through proton-boron nuclear reactions would be sufficient to enhance the biological effectiveness of the proton beams.We measured clonogenic survival in DU145 cells treated with 80.4-ppm BSH or 86.9-ppm BPA, or their respective vehicles, after irradiation with 6-MV X-rays, 1.2-keV/μm (low linear energy transfer [LET]) protons, or 9.9-keV/μm (high-LET) protons. We also measured γH2AX and 53BP1 foci in treated cells at 1 and 24 h after irradiation with the same conditions.We found that BSH radiosensitized DU145 cells across all radiation types. However, no difference was found in relative radiosensitization, characterized by the sensitization enhancement ratio or the relative biological effectiveness, for vehicle- versus BSH-treated cells. No differences were found in numbers of γH2AX or 53BP1 foci or γH2AX/53BP1 colocalized foci for vehicle- versus BSH-treated cells across radiation types. BPA did not radiosensitize DU145 cells nor induced any significant differences when comparing vehicle- versus BPA-treated cells for clonogenic cell survival or γH2AX and 53BP1 foci or γH2AX/53BP1 colocalized foci.Treatment with

Purpose/Objective(s) Radiotherapy (RT) has been shown, albeit rarely, to stimulate anti-tumor immune responses. Recent evidence suggests this can, in part, be attributed to the accumulation of cytoplasmic DNA in the form of micronuclei (MN) following RT, which can stimulate IRF3 and NF-κB immune signaling that recruits various immune components to the tumor microenvironment. We hypothesized this effect is increased by combining RT with AZD6738, an inhibitor of ATR, which is a key protein in DNA repair and cell cycle regulation. Furthermore, we propose that the more clustered DNA damage caused by protons results in a higher level of MN than photons when these radiation types are combined with AZD6738. Materials/Methods H1299 and PANC-1 cells were treated with 6 MV x-rays and 9.9 keV/mm (dose-weighted LET in water) protons alone or with AZD6738 (1 µM) to assess DNA repair, cell cycle distribution, MN and c-GAS positive MN (MN-cGAS+) 1 to 72 h after RT+AZD6738. We then irradiated a syngeneic 4T1 tumor model in BALB/c (hind leg tumors) with 6 MV x-rays to 3 × 6 Gy fractions with and without AZD6738 to assess tumor growth delay and the status of tumor infiltrating immune cells and immune cells in the draining lymph nodes (DLN). Results Protons+AZD6738 and photons+AZD6738 increased gH2AX foci, abrogated G2 arrest and amplified MN and MN-cGAS+ compared to photons or protons alone for both cell lines (H1299 and PANC-1). Compared to photons or AZD6738 alone, photons+AZD6738 delayed tumor growth, increased survival time and decreased the number of metastatic lung nodules. Photons+AZD6738 and photons alone increased T-regs and decreased T helper 1 cells in the tumor compared to vehicle and ATRi alone. Photons+AZD6738 increased and decreased CD8+ T cells in the tumor and DLN compared to the other treatments, respectively. Photons alone increased CD8+ PD-1+ T cells and photons+AZD6738 decreased that population back to baseline for both tumor and DLN. CD8+ GranzB+ T cells increased for photons+AZD6738 and photons alone in the tumor. Photons+AZD6738 and AZD6738 alone increased type 1 macrophages (M1) and decreased M2. Photons+AZD6738 increased the total population of dendritic cells (DCs) and non-significantly increased type 1 and 2 classical DCs compared to other treatments. Conclusion Our data show that RT+AZD6738 initiates intracellular immune signaling, likely attributable to disruption of DNA repair and abrogation of cell cycle arrest. We also observed this effect was amplified with proton RT. Our findings of increased immune signaling were corroborated in vivo with the observation that RT+AZD6738 increased CD4+, CD8+ and macrophage populations within the tumor, however after an initial delay, tumors continued to progress. Additional investigation is warranted to determine the balance of regulatory elements such as T-regs, immune checkpoints, and antitumor immune signaling.

PurposeTo develop an empirical model to predict radiosensitivity and relative biological effectiveness (RBE) after helium (He) and carbon (C) ion irradiation with or without DNA repair inhibitors.MethodsWe characterized survival in eight human cancer cell lines exposed to 6 MV photons and to He- and C-ions with linear energy transfer (LET) values of 2.2-60.5 keV/μm to verify that the radiosensitivity parameters (D5%, D10%, D20%, D37%, D50% and SF2Gy) correlate linearly between photon and ion radiation with or without DNA-PKcs or ATR inhibitors. Then, we parameterized the LET response of the parameters governing these linear correlations up to LET values of 225 keV/μm using the data in the Particle Irradiation Data Ensemble (PIDE) v3.2 database, creating a model that predicts a cell’s ion radiosensitivity, RBE and ion survival curve for a given LET on the basis of the cell’s photon radiosensitivity. We then trained this model using the PIDE database as a training dataset, and validated it by predicting the radiosensitivity of the cell lines we exposed to He- and C- ions with LET ranging from 2.2-60.5 keV/μm.ResultsRadiosensitivity to ions depended linearly with radiosensitivity of photons in the range of investigated LET values and the slopes and intercepts of these linear relationships within the PIDE database vary exponentially and linearly, respectively. Our model predicted ion radiosensitivity (e.g., D10%) within 5.1–21.3%, RBED10% within 5.0-17.1%, and ion mean inactivation dose within 6.7-25.1% for He- and C-ion LET ranging from 2.2-60.5 keV/μm.ConclusionsRadiosensitivity to He- and C-ions depend linearly with radiosensitivity to photons and can be used to predict ion radiosensitivity, RBE and cell survival curves for clinically relevant LET values from 2.2–60.5 keV/μm, with or without drug treatment.SUMMARYWe present a new empirical model capable of predicting clonogenic cell survival of cell lines exposed to helium and carbon ion beams. Our model is based on an observed linear correlation between radiosensitivity to ions and photons across a wide range of LET values. This linear correlation can be used to predict ion RBE, radiosensitivity, and the cell survival curve for a given LET all based on a cell’s photon survival curve.

PurposeTo show that radiation response across cancer cell lines of the same anatomic site and histologic type varies remarkably for protons and carbon (C) ions.Materials and MethodsWe measured and obtained from the literature clonogenic survival of human cancer cell lines of the lung (n=18), brain (n=10) and pancreas (n=10) exposed to photons, protons, and C-ions to assess their variability in response. We also treated cancer cell lines with DNA repair inhibitors prior to irradiation to assess how DNA repair capacity affects their variability in response. We quantified the variability in response by calculating the relative range (range/mean) and the coefficient of variation (COV) of the dose at 10% survival fraction (D10%) and relative biological effectiveness (RBE10%).ResultsThe relative range of D10% for lung cancer cell lines varied from 55-92% for photons, protons, and C-ions, with the relative range in RBE varying from 16-45% for protons and C-ions. For brain and pancreatic cancer cell lines, the relative range of D10% varied from 95-112%, and 39-75%, respectively, with the relative range in RBE varying from 27-33% and 25-50%, respectively. However, the COVs in D10% were approximately equal across radiation qualities, varying from 0.24±0.07–0.35±0.10, 0.35±0.09–0.69±0.62 and 0.13±0.03– 0.21±0.04 for lung, brain and pancreatic cancer cell lines, respectively. Greater relative ranges in D10% were observed in the cell lines with inhibited DNA repair, varying from 108%-157% for photons, protons, and C-ions, with relative ranges in RBE varying from 29-67%. The COVs in the D10% were also greater for the cell lines treated with inhibitors of DNA repair, varying from 0.34±0.09–0.41±0.06.ConclusionCell lines of the same anatomic site and histologic type have a remarkable variability in response, not only to photons but also to protons and C-ions. We attributed this variability to differences in DNA repair capacity.CategoryBiological Physics and Response Prediction

Purpose/Objective(s) Radiotherapy (RT) controls tumors primarily by inducing DNA damage. However, some tumor cells can repair RT-induced DNA damage and are thus radioresistant. RT could be made more effective by combining it with inhibitors of DNA damage response proteins (DDRi) to selectively render tumor cells more susceptible to RT-induced DNA damage. There are several DDRi currently approved by the FDA or in clinical trials for use as monotherapy, including inhibitors of DNA-PKcs, PARP, Rad 51, ATR and ATM. These proteins function in non-homologous end-joining (classical and alternative), homologous recombination, base excision repair or cell cycle checkpoint and disrupting any of which may enhance RT effectiveness. Because protons produce much more complex DNA lesion compared to x-rays, protons may amplify the effect of DDRis and further enhance potential activation of anti-tumor immune signaling. Our hypothesis is that DDRis sensitize cancer cells to RT and the sensitization effect is amplified by protons more so than x-rays with an enhancement in upstream immune signaling. Materials/Methods We used 4 cancer cell lines: H460, H1299, PANC-1 and Panc 10.05 and 5 DDRis targeting DNA-PKcs (NU7441), PARP (AZD2281), Rad 51 (B02), ATR (AZD6738) and ATM (KU55933) at various concentrations (0.1-10 µM). We irradiated cells with 6 MV x-rays or 9.9 keV/µm protons (dose-weighted LET in water) alone or with DDRis at various dose levels to assess cell survival via clonogenic assay, DNA damage via gH2AX and 53BP1 foci, micronuclei (MN) and cGAS-positive micronuclei (NM-cGAS+), the latter two being precursors of antitumor immunity. Results We observed that DDRis sensitized both x-rays and protons. Protons+ATMi (10 µM) sensitization relative to x-rays alone was increased 3.51-fold (± 0.09). The RBE ranged from 0.79 ± 0.03 to 1.54 ± 0.09 and were negatively correlated with x-rays sensitivity (r = -0.6160), suggesting that the greater the DDRi-induced sensitization, the less benefit there will be from the proton LET effect. Nonetheless, the sensitization relative to x-rays alone from combining DDRi with protons was generally larger (30 ± 3% on average). Generally, the combination of DDRi with RT increased gH2AX and 53BP1 foci at 24 h after radiation, and MN and MN-cGAS+ at 24 and 72 h after radiation with protons+DDRi having a higher increase relative to x-rays alone than x-rays+DDRi. Conclusion Inhibition of DNA-PKcs, PARP, Rad 51, ATR and ATM substantially sensitized several cancer cell lines to x-rays and protons. Although protons+DDRi reduced proton RBE, the inhibitor-induced sensitization outweighs the RBE loss, resulting in a net sensitization beyond x-rays+DDRi. The increased MN number indicates that pairing RT with DDRis may provide an additional benefit of antitumor immune stimulation. Additional preclinical work is warranted to elucidate the complex interaction of DDRi and RT in vivo.

Purpose High energetic carbon (C-) ion beams undergo nuclear interactions with tissue, producing secondary nuclear fragments. Thus, at depth, C-ion beams are composed of a mixture of different particles with different linear energy transfer (LET) values. We developed a technique to enable isolation of DNA damage response (DDR) in mixed radiation fields using beam line microscopy coupled with fluorescence nuclear track detectors (FNTDs). Methods We imaged live cells on a coverslip made of FNTDs right after C-ion, proton or photon irradiation using an in-house built confocal microscope placed in the beam path. We used the FNTD to link track traversals with DNA damage and separated DNA damage induced by primary particles from fragments. Results We were able to spatially link physical parameters of radiation tracks to DDR in live cells to investigate spatiotemporal DDR in multi-ion radiation fields in real time, which was previously not possible. We demonstrated that the response of lesions produced by the high-LET primary particles associates most strongly with cell death in a multi-LET radiation field, and that this association is not seen when analyzing radiation induced foci in aggregate without primary/fragment classification. Conclusions We report a new method that uses confocal microscopy in combination with FNTDs to provide submicrometer spatial-resolution measurements of radiation tracks in live cells. Our method facilitates expansion of the radiation-induced DDR research because it can be used in any particle beam line including particle therapy beam lines. Category Biological Physics and Response Prediction.

Purpose This study seeks to identify biological factors that may yield a therapeutic advantage of proton therapy versus photon therapy. Specifically, we address the role of nonhomologous end-joining (NHEJ) and homologous recombination (HR) in the survival of cells in response to clinical photon and proton beams. Methods and Materials We irradiated HT1080, M059K (DNA-PKcs+/+), and HCC1937 human cancer cell lines and their isogenic counterparts HT1080-shDNA-PKcs, HT1080-shRAD51IND, M059J (DNA-PKcs–/–), and HCC1937-BRCA1 (BRCA1 complemented) to assess cell clonogenic survival and γ-H2AX radiation-induced foci. Cells were irradiated with either clinically relevant photons or 1 of 3 proton linear energy transfer (LET) values. Results Our results indicate that NHEJ deficiency is more important in dictating cell survival than proton LET. Cells with disrupted HR through BRCA1 mutation showed increased radiosensitivity only for high-LET protons whereas RAD51 depletion showed increased radiosensitivity for both photons and protons. DNA double strand breaks, assessed by γ-H2AX radiation-induced foci, showed greater numbers after 24 hours in cells exposed to higher LET protons. We also observed that NHEJ-deficient cells were unable to repair the vast majority of double strand breaks after 24 hours. Conclusions BRCA1 mutation significantly sensitizes cells to protons, but not photons. Loss of NHEJ renders cells hypersensitive to radiation, whereas the relative importance of HR increases with LET across several cell lines. This may be attributable to the more clustered damage induced by higher LET protons, which are harder to repair through NHEJ. This highlights the importance of tumor biology in dictating treatment modality and suggests BRCA1 as a potential biomarker for proton therapy response. Our data also support the use of pharmacologic inhibitors of DNA repair to enhance the sensitivity to different radiation types, although this raises issues for normal tissue toxicity.

In this work we characterized the dose and linear energy transfer (LET) (ionization density) dependence of commercial Al 2 O 3 :C optically stimulated luminescence (OSL) detectors (OSLDs) exposed to clinical photon and proton beams. We characterized the dose-dependence of the OSL signal, OSL curve shape, and the relative intensities of the blue and ultraviolet (UV) OSL emission bands using different readout protocols and beam qualities. We irradiated OSLDs with absorbed doses ranging from 0.1 Gy to 100 Gy in a 6-MV photon beam and from 0.1 Gy to 50 Gy in 140- and 250-MeV proton beams. Readouts were done with both continuous-wave (CWOSL) and pulsed (POSL) stimulation. The linearity of the OSLD dose–response depended on readout protocol and radiation type. Improved linearity was found for OSLDs irradiated with beams of increased LET, and for OSL signals containing only the blue emission band of Al 2 O 3 :C (which remained linear for doses up to 10 Gy for 140-MeV proton beam irradiations). The OSL curve shape did not vary with dose in the low-dose region (below 5 Gy depending on readout protocol), but beyond this, curves decayed more rapidly with increasing dose. Similarly, the ratio of blue to UV emission band intensities in the OSL signal did not vary with dose up to 5 Gy (depending on readout protocol), beyond which the ratio decreased with increasing dose. Because both the OSL curve shape and the ratio of blue to UV emission intensities have been investigated as potential parameters for measurements of LET, the constancy of these two quantities at doses relevant to radiotherapy is encouraging for the potential development of novel OSL methods to measure LET. Our findings are expected to contribute to the development of (i) improved readout protocols for commercially available Al 2 O 3 :C OSLDs and (ii) methods to measure radiation quality and LET.

Purpose Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. Methods and Materials We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10 6 protons/cm 2 . Results We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. Conclusions This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.

Purpose: To construct a custom and portable fluorescence confocal laser-scanning microscope (FCLSM) that can be placed in the path of therapeutic radiation beams to study real-time radiation-induced damage response in live cells. Methods: We designed and constructed a portable FCLSM with three laser diodes for excitation (405, 488, and 635 nm). An objective lens focuses the excitation light and collects fluorescence from the sample. A pair of galvanometer mirrors scans/collects the laser beam/fluorescence along the focal plane (x/y-directions). A stepper motor stage scans in the axial direction and positions the x/y of the image field. Barrier filters and dichroic mirrors are used to route the spectral emission bands to the appropriate photodetector. An avalanche photodiode collects near-infrared fluorescence; a photodiode collects back-reflected 635 nm light; and a photomultiplier tube collects green fluorescence in the range of eGFP/eYFP. A 200-µm diameter pinhole was used to implement the confocal geometry for near-infrared and red channels and a 150-µm diameter pinhole for the green channel. Data acquisition and system control were achieved using a high-throughput data acquisition card. In-house software developed in LabVIEW was used to control the hardware, collect data from the photodetectors and reconstruct the confocal images. Results: 6 frames/s can be acquired for a 25 µm2 (128×128 pixels) field of view, visualizing the entire volume of the cell nucleus (∼10 µm depth) in

Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

Purpose: To characterize the response of Al{sub 2}O{sub 3}:C optically stimulated luminescence (OSL) detectors (OSLDs) exposed to therapeutic proton beams of differing beam quality. Methods: We prepared Al{sub 2}O{sub 3}:C OSLDs from the same material as commercially available nanoDot dosimeters (Landauer, Inc). We irradiated the OSLDs in modulated proton beams of varying quality, as defined by the residual range. An absorbed dose to water of 0.2 Gy was delivered to all OSLDs with the residual range values varying from 0.5 to 23.5 cm (average LET in water from ∼0.5 to 2.5 keV/µm). To investigate the beam quality dependence of different emission bands within the OSL spectrum, we performed OSLD readouts using both continuous-wave stimulation (CW-OSL) and pulsed stimulation (P-OSL) with two sets of optical filters (Hoya U-340 and Kopp 5113). For all readout modes, the relative absorbed dose sensitivity (S{sub rel}) for each beam quality was calculated using OSLDs irradiated in a 6 MV photon beam as a reference. Results: We found that the relative absorbed dose sensitivity was highly dependent on both readout mode and integration time of the OSL signal. For CW-OSL signals containing only the blue emission band, S{sub rel} was between 0.85 and 0.94 for 1more » s readouts and between 0.82 and 0.93 for 10 s readouts. Similarly, for P-OSL readouts containing only the blue emission band S{sub rel} ranged from 0.86 to 0.91, and 0.82 to 0.93 for 1 s and 10 s readouts, respectively. For OSLD signals containing only the UV emission band, S{sub rel} ranged from 1.00 to 1.46, and 0.97 to 1.30 for P-OSL readouts of 1 s and 10 s, respectively. Conclusion: For measurements of absorbed dose using Al{sub 2}O{sub 3}:C OSLDs in therapeutic proton beams, dependence on beam quality was smallest for readout protocols that selected the blue emission band with small integration times. DA Granville received financial support from the Natural Sciences and Engineering Research Council of Canada.« less

Purpose: To determine the effect of a strong magnetic field on TLD-100, OSLD (Al2O2:C), and PRESAGE dosimetry devices. This study will help to determine which types of dosimeters can be used for quality assurance and in-vivo dosimetry measurements in a magnetic resonance imaginglinear accelerator (MRI-linac) system. Methods: The dosimeters were separated into two categories which were either exposed or not exposed to a strong magnetic field. In each category a set of dosimeters was irradiated with 0, 2, or 6 Gy. To expose the dosimeters to a magnetic field the samples in that category were place in a Bruker small animal magnetic resonance scanner at a field strength slightly greater than 2.5 T for at least 1 hour preirradiation and at least 1 hour post-irradiation. Irradiations were performed with a 6 MV x-ray beam from a Varian TrueBeam linac with 10×10 cm2 field at a 600 MU/min dose rate. The samples that received no radiation dose were used as control detectors. Results: The readouts of the dosimeters which were not exposed to a strong magnetic field were compared with the measurements of the dosimetry devices which were exposed to a magnetic field. No significant differences (less than 2% difference) in the performance of TLD, OSLD, or PRESAGE dosimeters due to exposure to a strong magnetic field were observed. Conclusion: Exposure to a strong magnetic field before and after irradiation does not appear to change the dosimetric properties of TLD, OSLD, or PRESAGE which indicates that these dosimeters have potential for use in quality assurance and in-vivo dosimetry in a MRI-linac. We plan to further test the effect of magnetic fields on these devices by irradiating them in the presence of a magnetic fields similar to those produced by a MRI-linac system. Elekta-MD Anderson Cancer Center Research Agreement