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The use of charged particle radiotherapy is currently increasing, but combination therapy with DNA repair inhibitors remains to be exploited in the clinic. The high-linear energy transfer (LET) radiation delivered by charged particles causes clustered DNA damage, which is particularly effective in destroying cancer cells. Whether the DNA damage response to this type of damage is different from that elicited in response to low-LET radiation, and if and how it can be targeted to increase treatment efficacy, is not fully understood. Although several preclinical studies have reported radiosensitizing effects when proton or carbon ion irradiation is combined with inhibitors of, e.g., PARP, ATR, ATM, or DNA-PKcs, further exploration is required to determine the most effective treatments. Here, we examine what is known about repair pathway choice in response to high- versus low-LET irradiation, and we discuss the effects of inhibitors of these pathways when combined with protons and carbon ions. Additionally, we explore the potential effects of DNA repair inhibitors on antitumor immune signaling upon proton and carbon ion irradiation. Due to the reduced effect on healthy tissue and better immune preservation, particle therapy may be particularly well suited for combination with DNA repair inhibitors.
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Dano ao DNA , Reparo do DNA , Radioterapia com Íons Pesados , Terapia com Prótons , Humanos , Reparo do DNA/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Animais , Transferência Linear de EnergiaRESUMO
Introduction: Inhibitors of the ATR kinase act as radiosensitizers through abrogating the G2 checkpoint and reducing DNA repair. Recent studies suggest that ATR inhibitors can also increase radiation-induced antitumor immunity, but the underlying immunomodulating mechanisms remain poorly understood. Moreover, it is poorly known how such immune effects relate to different death pathways such as caspase-dependent apoptosis. Here we address whether ATR inhibition in combination with irradiation may increase the presentation of hallmark factors of immunogenic cell death (ICD), and to what extent caspase activation regulates this response. Methods: Human lung cancer and osteosarcoma cell lines (SW900, H1975, H460, U2OS) were treated with X-rays and ATR inhibitors (VE822; AZD6738) in the absence and presence of a pan-caspase inhibitor. The ICD hallmarks HMGB1 release, ATP secretion and calreticulin surface-presentation were assessed by immunoblotting of growth medium, the CellTiter-Glo assay and an optimized live-cell flow cytometry assay, respectively. To obtain accurate measurement of small differences in the calreticulin signal by flow cytometry, we included normalization to a barcoded control sample. Results: Extracellular release of HMGB1 was increased in all the cell lines at 72 hours after the combined treatment with radiation and ATR inhibitors, relative to mock treatment or cells treated with radiation alone. The HMGB1 release correlated largely - but not strictly - with loss of plasma membrane integrity, and was suppressed by addition of the caspase inhibitor. However, one cell line showed HMGB1 release despite caspase inhibition, and in this cell line caspase inhibition induced pMLKL, a marker for necroptosis. ATP secretion occurred already at 48 hours after the co-treatment and did clearly not correlate with loss of plasma membrane integrity. Addition of pan-caspase inhibition further increased the ATP secretion. Surface-presentation of calreticulin was increased at 24-72 hours after irradiation, but not further increased by either ATR or caspase inhibition. Conclusion: These results show that ATR inhibition can increase the presentation of two out of three ICD hallmark factors from irradiated human cancer cells. Moreover, caspase activation distinctly affects each of the hallmark factors, and therefore likely plays a dual role in tumor immunogenicity by promoting both immunostimulatory and -suppressive effects.
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Caspases , Proteína HMGB1 , Humanos , Caspases/metabolismo , Proteína HMGB1/metabolismo , Calreticulina/metabolismo , Inibidores de Caspase , Morte Celular Imunogênica , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases , Trifosfato de Adenosina , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
PURPOSE: Radiation-induced activation of cell cycle checkpoints have been of long-standing interest. The WEE1, CHK1 and ATR kinases are key factors in cell cycle checkpoint regulation and are essential for the S and G2 checkpoints. Here, we review the rationale for why inhibitors of WEE1, CHK1 and ATR could be beneficial in combination with radiation. CONCLUSIONS: Combined treatment with radiation and inhibitors of these kinases results in checkpoint abrogation and subsequent mitotic catastrophe. This might selectively radiosensitize tumor cells, as they often lack the p53-dependent G1 checkpoint and therefore rely more on the G2 checkpoint to repair DNA damage. Further affecting the repair of radiation damage, inhibition of WEE1, CHK1 or ATR also specifically suppresses the homologous recombination repair pathway. Moreover, inhibition of these kinases can induce massive replication stress during S phase of the cell cycle, likely contributing to eliminate radioresistant S phase cells. Intriguingly, recent findings suggest that cell cycle checkpoint inhibitors in combination with radiation can also enhance anti-tumor immune effects. Altogether, the expanding knowledge about the functional roles of WEE1, CHK1 and ATR inhibitors support that they are promising candidates for use in combination with radiation treatment.
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Proteínas Tirosina Quinases , Radioterapia (Especialidade) , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/metabolismo , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Linhagem Celular Tumoral , Proteínas Mutadas de Ataxia Telangiectasia/genéticaRESUMO
Recent studies suggest that inhibition of the ATR kinase can potentiate radiation-induced antitumor immune responses, but the extent and mechanisms of such responses in human cancers remain scarcely understood. We aimed to assess whether the ATR inhibitors VE822 and AZD6738, by abrogating the G2 checkpoint, increase cGAS-mediated type I IFN response after irradiation in human lung cancer and osteosarcoma cell lines. Supporting that the checkpoint may prevent IFN induction, radiation-induced IFN signaling declined when the G2 checkpoint arrest was prolonged at high radiation doses. G2 checkpoint abrogation after co-treatment with radiation and ATR inhibitors was accompanied by increased radiation-induced IFN signaling in four out of five cell lines tested. Consistent with the hypothesis that the cytosolic DNA sensor cGAS may detect DNA from ruptured micronuclei after G2 checkpoint abrogation, cGAS co-localized with micronuclei, and depletion of cGAS or STING abolished the IFN responses. Contrastingly, one lung cancer cell line showed no increase in IFN signaling despite irradiation and G2 checkpoint abrogation. This cell line showed a higher level of the exonuclease TREX1 than the other cell lines, but TREX1 depletion did not enhance IFN signaling. Rather, addition of a pan-caspase inhibitor restored the IFN response in this cell line and also increased the responses in the other cell lines. These results show that treatment-induced caspase activation can suppress the IFN response after co-treatment with radiation and ATR inhibitors. Caspase activation thus warrants further consideration as a possible predictive marker for lack of IFN signaling.
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Intraoperative image-guided surgical navigation for craniospinal procedures has significantly improved accuracy by providing an avenue for the surgeon to visualize underlying internal structures corresponding to the exposed surface anatomy. Despite the obvious benefits of surgical navigation, surgeon adoption remains relatively low due to long setup and registration times, steep learning curves, and workflow disruptions. We introduce an experimental navigation system utilizing optical topographical imaging (OTI) to acquire the 3D surface anatomy of the surgical cavity, enabling visualization of internal structures relative to exposed surface anatomy from registered preoperative images. Our OTI approach includes near instantaneous and accurate optical measurement of >250,000 surface points, computed at >52,000 points-per-second for considerably faster patient registration than commercially available benchmark systems without compromising spatial accuracy. Our experience of 171 human craniospinal surgical procedures, demonstrated significant workflow improvement (41 s vs. 258 s and 794 s, p < 0.05) relative to benchmark navigation systems without compromising surgical accuracy. Our advancements provide the cornerstone for widespread adoption of image guidance technologies for faster and safer surgeries without intraoperative CT or MRI scans. This work represents a major workflow improvement for navigated craniospinal procedures with possible extension to other image-guided applications.
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Encéfalo , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional , Imageamento por Ressonância Magnética/métodos , Medula Espinal , Cirurgia Assistida por Computador , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/cirurgia , Humanos , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Curva de Aprendizado , Neurocirurgiões/educação , Medula Espinal/diagnóstico por imagem , Medula Espinal/cirurgia , Cirurgia Assistida por Computador/instrumentação , Cirurgia Assistida por Computador/métodos , SuínosRESUMO
Speckle statistics of flowing scatterers have been well documented in the literature. Speckle variance optical coherence tomography exploits the large variance values of intensity changes in time caused mainly by the random backscattering of light resulting from translational activity of red blood cells to map out the microvascular networks. A method to map out the microvasculature malformation of skin based on the time-domain histograms of individual pixels is presented with results obtained from both normal skin and skin containing vascular malformation. Results demonstrated that this method can potentially map out deeper blood vessels and enhance the visualization of microvasculature in low signal regions, while being resistant against motion (e.g., patient tremor or internal reflex movements). The overall results are manifested as more uniform en face projection maps of microvessels. Potential applications include clinical imaging of skin vascular abnormalities and wide-field skin angiography for the study of complex vascular networks.
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Angiografia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microvasos/fisiopatologia , Pele/fisiopatologia , Telangiectasia Hemorrágica Hereditária/diagnóstico , Telangiectasia Hemorrágica Hereditária/fisiopatologia , Tomografia de Coerência Óptica/métodos , Angiografia/instrumentação , Animais , Velocidade do Fluxo Sanguíneo , Interpretação Estatística de Dados , Humanos , Aumento da Imagem/métodos , Imagens de Fantasmas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Pele/irrigação sanguínea , Tomografia de Coerência Óptica/instrumentaçãoRESUMO
In this work, we explored the potential of measuring shear wave propagation using optical coherence elastography (OCE) in an inhomogeneous phantom and carotid artery samples based on a swept-source optical coherence tomography (OCT) system. Shear waves were generated using a piezoelectric transducer transmitting sine-wave bursts of 400 µs duration, applying acoustic radiation force (ARF) to inhomogeneous phantoms and carotid artery samples, synchronized with a swept-source OCT (SS-OCT) imaging system. The phantoms were composed of gelatin and titanium dioxide whereas the carotid artery samples were embedded in gel. Differential OCT phase maps, measured with and without the ARF, detected the microscopic displacement generated by shear wave propagation in these phantoms and samples of different stiffness. We present the technique for calculating tissue mechanical properties by propagating shear waves in inhomogeneous tissue equivalent phantoms and carotid artery samples using the ARF of an ultrasound transducer, and measuring the shear wave speed and its associated properties in the different layers with OCT phase maps. This method lays the foundation for future in-vitro and in-vivo studies of mechanical property measurements of biological tissues such as vascular tissues, where normal and pathological structures may exhibit significant contrast in the shear modulus.
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Temperature-compensated 3D fiber shape sensing is demonstrated with femtosecond laser direct-written optical and Bragg grating waveguides that were distributed axially and radially inside a single coreless optical fiber. Efficient light coupling between the laser-written optical circuit elements and a standard single-mode fiber (SMF) was obtained for the first time by 3D laser writing of a 1 × 3 directional coupler to meet with the core waveguide in the fusion-spliced SMF. Simultaneous interrogation of nine Bragg gratings, distributed along three laterally offset waveguides, is presented through a single waveguide port at 1 kHz sampling rate to follow the Bragg wavelength shifts in real-time and thereby infer shape and temperature profile unambiguously along the fiber length. This distributed 3D strain and thermal sensor is freestanding, flexible, compact, lightweight and opens new directions for creating fiber cladding photonic devices for a wide range of applications from shape and thermal sensing to guidance of biomedical catheters and tools in minimally invasive surgery.
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High-resolution mapping of microvasculature has been applied to diverse body systems, including the retinal and choroidal vasculature, cardiac vasculature, the central nervous system, and various tumor models. Many imaging techniques have been developed to address specific research questions, and each has its own merits and drawbacks. Understanding, optimization, and proper implementation of these imaging techniques can significantly improve the data obtained along the spectrum of unique research projects to obtain diagnostic clinical information. We describe the recently developed algorithms and applications of two general classes of microvascular imaging techniques: speckle-variance and phase-variance optical coherence tomography (OCT). We compare and contrast their performance with Doppler OCT and optical microangiography. In addition, we highlight ongoing work in the development of variance-based techniques to further refine the characterization of microvascular networks.
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Processamento de Imagem Assistida por Computador , Microvasos/anatomia & histologia , Tomografia de Coerência Óptica , Algoritmos , Animais , HumanosRESUMO
Feasibility of detecting intravascular flow using a catheter based endovascular optical coherence tomography (OCT) system is demonstrated in a porcine carotid model in vivo. The effects of A-line density, radial distance, signal-to-noise ratio, non-uniform rotational distortion (NURD), phase stability of the swept wavelength laser and interferometer system on Doppler shift detection limit were investigated in stationary and flow phantoms. Techniques for NURD induced phase shift artifact removal were developed by tracking the catheter sheath. Detection of high flow velocity (~51 cm/s) present in the porcine carotid artery was obtained by phase unwrapping techniques and compared to numerical simulation, taking into consideration flow profile distortion by the eccentrically positioned imaging catheter. Using diluted blood in saline mixture as clearing agent, simultaneous Doppler OCT imaging of intravascular flow and structural OCT imaging of the carotid artery wall was feasible. To our knowledge, this is the first in vivo demonstration of Doppler imaging and absolute measurement of intravascular flow using a rotating fiber catheter in carotid artery.
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Advances in swept source laser technology continues to increase the imaging speed of swept-source optical coherence tomography (SS-OCT) systems. These fast imaging speeds are ideal for microvascular detection schemes, such as speckle variance (SV), where interframe motion can cause severe imaging artifacts and loss of vascular contrast. However, full utilization of the laser scan speed has been hindered by the computationally intensive signal processing required by SS-OCT and SV calculations. Using a commercial graphics processing unit that has been optimized for parallel data processing, we report a complete high-speed SS-OCT platform capable of real-time data acquisition, processing, display, and saving at 108,000 lines per second. Subpixel image registration of structural images was performed in real-time prior to SV calculations in order to reduce decorrelation from stationary structures induced by the bulk tissue motion. The viability of the system was successfully demonstrated in a high bulk tissue motion scenario of human fingernail root imaging where SV images (512 × 512 pixels, n = 4) were displayed at 54 frames per second.
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Optical coherence tomography (OCT) has the combined advantage of high temporal (µsec) and spatial (<10µm) resolution. These features make it an attractive tool to study the dynamic relationship between neural activity and the surrounding blood vessels in the spinal cord, a topic that is poorly understood. Here we present work that aims to optimize an in vivo OCT imaging model of the rodent spinal cord. In this study we image the microvascular networks of both rats and mice using speckle variance OCT. This is the first report of depth resolved imaging of the in vivo spinal cord using an entirely endogenous contrast mechanism.
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In this work, we explored the potential of measuring shear wave propagation using optical coherence elastography (OCE) based on a swept-source optical coherence tomography (OCT) system. Shear waves were generated using a 20 MHz piezoelectric transducer (circular element 8.5 mm diameter) transmitting sine-wave bursts of 400 µs, synchronized with the OCT swept source wavelength sweep. The acoustic radiation force (ARF) was applied to two gelatin phantoms (differing in gelatin concentration by weight, 8% vs. 14%). Differential OCT phase maps, measured with and without the ARF, demonstrate microscopic displacement generated by shear wave propagation in these phantoms of different stiffness. We present preliminary results of OCT derived shear wave propagation velocity and modulus, and compare these results to rheometer measurements. The results demonstrate the feasibility of shear wave OCE (SW-OCE) for high-resolution microscopic homogeneous tissue mechanical property characterization.
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Quantum dot (QD) contrast-enhanced molecular imaging has potential for early cancer detection and image guided treatment, but there is a lack of quantitative image contrast data to determine optimum QD administered doses, affecting the feasibility, risk and cost of such procedures, especially in vivo. Vascular fluorescence contrast-enhanced imaging is performed on nude mice bearing dorsal skinfold window chambers, injected with 4 different QD solutions emitting in the visible and near infrared. Linear relationships are observed among the vascular contrast, injected contrast agent volume, and QD concentration in blood. Due primarily to differential light absorption by blood, the vasculature is optimally visualized when exciting in the 435-480 nm region in 81% of the cases (89 out of 110 regions of interest in 22 window chambers). The threshold dose, defined here as the quantity of injected nanoparticles required to yield a vascular target-to-autofluorescence ratio of 2, varies from 10.6 to 0.15 pmol g(-1) depending on the QD emission wavelength. The wavelength optimization maximum and broadband gain, defined as the ratio of threshold doses estimated for optimal and suboptimal (worst wavelength or broadband) spectral illumination, has average values of 4.5 and 1.9, respectively. This study demonstrates, for the first time, optimized QD imaging in vivo. It also proposes and validates a theoretical framework for QD dose estimation and quantifies the effects of blood absorption, QD emission wavelength, and vessel diameter relative to the threshold dose.
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Imagem Molecular/métodos , Pontos Quânticos , Animais , Feminino , Fluorescência , Humanos , Camundongos , Espectrometria de FluorescênciaRESUMO
Application of speckle variance optical coherence tomography (OCT) to endovascular imaging faces difficulty of extensive motion artifacts inherently associated with arterial pulsations in addition to other physiological movements. In this study, we employed a technique involving a fourth order statistical method, kurtosis, operating on the endovascular OCT intensity images to visualize the vasa vasorum of carotid artery in vivo and identify its flow dynamic in a porcine model. The intensity kurtosis technique can distinguish vasa vasorum from the surrounding tissues in the presence of extensive time varying noises and dynamic motions of the arterial wall. Imaging of vasa vasorum and its proliferation, may compliment the growing knowledge of structural endovascular OCT in assessment and treatment of atherosclerosis in coronary and carotid arteries.
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A dynamic light scattering technique is implemented using optical coherence tomography (OCT) to measure the change in intracellular motion as cells undergo apoptosis. Acute myeloid leukemia cells were treated with cisplatin and imaged at a frame rate of 166 Hz using a 1300 nm swept-source OCT system at various times over a period of 48 h. Time correlation analysis of the speckle intensities indicated a significant increase in intracellular motion 24 h after treatment. This rise in intracellular motion correlated with histological findings of irregularly shaped and fragmented cells indicative of cell membrane blebbing and fragmentation.
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Apoptose/fisiologia , Tomografia de Coerência Óptica/métodos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Luz , Fenômenos Ópticos , Espalhamento de Radiação , Tomografia de Coerência Óptica/estatística & dados numéricosRESUMO
A novel (to our knowledge) dual-core ytterbium (Yb(3+)) doped fiber, as an optically pumped amplifier, boosts the output power from a 1060 nm swept source laser beyond 250 mW, while providing a wavelength tuning range of 93 nm, for optical coherence tomography (OCT) imaging. The design of the dual-core Yb-doped fiber amplifier and its multiple wavelength optical pumping scheme to optimize output bandwidth are discussed. Use of the dual-core fiber amplifier showed no appreciable degradation to the coherence length of the seed laser. The signal intensity improvement of this amplifier is demonstrated on a multichannel in vivo OCT imaging system at 1060 nm.
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Tomografia de Coerência Óptica/instrumentação , Tomografia de Coerência Óptica/métodos , Itérbio/química , Animais , Xenopus laevis/anatomia & histologiaRESUMO
The transition from benchtop to clinical system often requires the medical technology to be robust, portable and accurate. This poses a challenge to current swept source optical coherence tomography imaging systems, as the bulk of the systems footprint is due to laser components. With the recent advancement of micromachining technology, we demonstrate the characterization of a microelectromechanical system (MEMS) swept source laser for optical coherence tomography imaging (OCT). This laser utilizes a 2 degree of freedom MEMS scanning mirror and a diffraction grating, which are arranged in a Littrow configuration. This resulted in a swept source laser that was capable of scanning at 23.165 kHz (bidirectional) or 11.582 kHz (unidirectional). The free spectral range of the laser was ≈ 100 nm with a central wavelength of ≈ 1330 nm. The 6 dB roll off depth was measured to be at 2.5 mm. Furthermore, the structural morphology of a human finger and tadpole (Xenopus laevis) were evaluated. The overall volumetric footprint of the laser source was measured to be 70 times less than non-MEMS swept sources. Continued work on the miniaturization of OCT system is on going. It is hypothesized that the overall laser size can be reduced for suitable OCT imaging for a point of care application.
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Embrião não Mamífero/metabolismo , Tecnologia de Fibra Óptica/métodos , Dedos/patologia , Sistemas Microeletromecânicos , Tomografia de Coerência Óptica/métodos , Animais , Desenho de Equipamento , Humanos , Lasers , Miniaturização , Fibras Ópticas , Espectrofotometria/métodos , Xenopus laevisRESUMO
We optimize speckle variance optical coherence tomography (svOCT) imaging of microvasculature in high and low bulk tissue motion scenarios. To achieve a significant level of image contrast, frame rates must be optimized such that tissue displacement between frames is less than the beam radius. We demonstrate that higher accuracy estimates of speckle variance can enhance the detection of capillaries. These findings are illustrated in vivo by imaging the dorsal window chamber model (low bulk motion). We also show svOCT imaging of the nonstabilized finger (high bulk motion), using optimized imaging parameters, demonstrating better vessel detection than Doppler OCT.
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Microvasos/citologia , Tomografia de Coerência Óptica/métodos , Gliossarcoma/patologia , Gliossarcoma/fisiopatologia , Humanos , Microvasos/patologia , Microvasos/fisiologia , Microvasos/fisiopatologia , Movimento , Unhas/irrigação sanguínea , Imagens de FantasmasRESUMO
We demonstrate the potential of a forward-looking Doppler optical coherence tomography (OCT) probe for color flow imaging in several commonly seen narrowed artery morphologies. As a proof of concept, we present imaging results of a surgically exposed thrombotic occlusion model that was imaged superficially to demonstrate that Doppler OCT can identify flow within the recanalization channels of a blocked artery. We present Doppler OCT images in which the flow is nearly antiparallel to the imaging direction. These images are acquired using a flexible 2.2-mm-diam catheter that used electrostatic actuation to scan up to 30 deg ahead of the distal end. Doppler OCT images of physiologically relevant flow phantoms consisting of small channels and tapered entrance geometries are demonstrated.