Potential Benefits of Combining Proton or Carbon Ion Therapy with DNA Damage Repair Inhibitors.

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.

Temperature-compensated fiber-optic 3D shape sensor based on femtosecond laser direct-written Bragg grating waveguides.

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.

Immunogenic cell death after combined treatment with radiation and ATR inhibitors is dually regulated by apoptotic caspases.

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.

Expanding roles of cell cycle checkpoint inhibitors in radiation oncology.

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.

Estimation of minimum doses for optimized quantum dot contrast-enhanced vascular imaging in vivo.

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.

Caspase activation counteracts interferon signaling after G2 checkpoint abrogation by ATR inhibition in irradiated human cancer cells.

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.

Dual-core ytterbium fiber amplifier for high-power 1060 nm swept source multichannel optical coherence tomography imaging.

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.

Optimized speckle variance OCT imaging of microvasculature.

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.

High-power wavelength-swept laser in Littman telescope-less polygon filter and dual-amplifier configuration for multichannel optical coherence tomography.

We report a high-power wavelength-swept laser source for multichannel optical coherence tomography (OCT) imaging. Wavelength tuning is performed by a compact telescope-less polygon-based filter in Littman arrangement. High output power is achieved by incorporating two serial semiconductor optical amplifiers in the laser cavity in Fourier domain mode-locked configuration. The measured wavelength tuning range of the laser is 111 nm centered at 1329 nm, coherence length of 5.5 mm, and total average output power of 131 mW at 43 kHz sweeping rate. Multichannel simultaneous OCT imaging at an equivalent A-scan rate of 258 kHz is demonstrated.

High power wavelength linearly swept mode locked fiber laser for OCT imaging.

We report a long coherence length, high power, and wide tuning range wavelength linearly swept fiber mode-locked laser based on polygon scanning filters. An output power of 52.6 mW with 112 nm wavelength tuning range at 62.6 kHz sweeping rate has been achieved. The coherence length is long enough to enable imaging over 8.1 mm depth when the sensitivity decreases by 8.7 dB (1/e(2)). The Fourier components are still distinguishable when the ranging depth exceeds 15 mm, which corresponds to 30 mm optical path difference in air. The parameters that are critical to OCT imaging quality such as polygon filter linewidth, the laser coherence length, output power, axial resolution and the Fourier sensitivity have been investigated theoretically and experimentally. Since the wavelength is swept linearly with time, an analytical approach has been developed for transforming the interference signal from equidistant spacing in wavelength to equidistant spacing in frequency. Axial resolution of 7.9 microm in air has been achieved experimentally that approaches the theoretical limit.

Intravital high-resolution optical imaging of individual vessel response to photodynamic treatment.

Intravital imaging using confocal microscopy facilitates high-resolution studies of cellular and molecular events in vivo. We use this, complemented by Doppler optical coherence tomography (OCT), to assess blood flow in a mouse dorsal skin-fold window chamber model to image the response of individual blood vessels to localized photodynamic therapy (PDT). Specific fluorescent cell markers were used to assess the effect on the vascular endothelial cell lining of the treated vessels. A fluorescently tagged antibody against an endothelial transmembrane glycoprotein (CD31) was used to image endothelial cell integrity in the targeted blood vessel. A cell permeability (viability) indicator, SYTOX Orange, was also used to further assess damage to endothelial cells. A fluorescently labeled anti-CD41 antibody that binds to platelets was used to confirm platelet aggregation in the treated vessel. These optical techniques enable dynamic assessment of responses to PDT in vivo, at both the vascular endothelial cell and whole vessel levels.

High Speed, High Density Intraoperative 3D Optical Topographical Imaging with Efficient Registration to MRI and CT for Craniospinal Surgical Navigation.

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.

Doppler optical cardiogram gated 2D color flow imaging at 1000 fps and 4D in vivo visualization of embryonic heart at 45 fps on a swept source OCT system.

We report a Doppler optical cardiogram gating technique for increasing the effective frame rate of Doppler optical coherence tomography (DOCT) when imaging periodic motion as found in the cardiovascular system of embryos. This was accomplished with a Thorlabs swept-source DOCT system that simultaneously acquired and displayed structural and Doppler images at 12 frames per second (fps). The gating technique allowed for ultra-high speed visualization of the blood flow pattern in the developing hearts of African clawed frog embryos (Xenopus laevis) at up to 1000 fps. In addition, four-dimensional (three spatial dimensions + temporal) Doppler imaging at 45 fps was demonstrated using this gating technique, producing detailed visualization of the complex cardiac motion and hemodynamics in a beating heart.

Interstitial Doppler optical coherence tomography monitors microvascular changes during photodynamic therapy in a Dunning prostate model under varying treatment conditions.

We measure the tumor vascular response to varying irradiance rates during photodynamic therapy (PDT) in a Dunning rat prostate model with interstitial Doppler optical coherence tomography (IS-DOCT). Rats are given a photosensitizer drug, Photofrin, and the tumors are exposed to light (635 nm) with irradiance rates ranging from 8 to 133 mWcm(2) for 25 min, corresponding to total irradiance of 12 to 200 Jcm(2) (measured at surface). The vascular index computed from IS-DOCT results shows the irradiance rate and total irradiance dependent microvascular shutdown in the tumor tissue during PDT. While faster rates of vascular shutdown were associated with increasing PDT irradiance rate and total irradiance, a threshold effect was observed as irradiance rates above 66 mWcm(2) (surface), where no further increase in vascular shutdown rate was detected. The maximum post-treatment vascular shutdown (81%) without immediate microcirculatory recovery was reached with the PDT condition of 33 mWcm(2) and 50 Jcm(2). Control groups without Photofrin show no significant microvascular changes. Microvascular shutdown occurs at different rates and shows correlation with PDT total irradiance and irradiance rates. These dependencies may play an important role in PDT treatment planning, feedback control for treatment optimization, and post-treatment assessment.

Optical coherence tomography detection of shear wave propagation in inhomogeneous tissue equivalent phantoms and ex-vivo carotid artery samples.

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.

Histogram flow mapping with optical coherence tomography for in vivo skin angiography of hereditary hemorrhagic telangiectasia.

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.

Review of speckle and phase variance optical coherence tomography to visualize microvascular networks.

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.

Feasibility of optical coherence elastography measurements of shear wave propagation in homogeneous tissue equivalent phantoms.

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.

In vivo feasibility of endovascular Doppler optical coherence tomography.

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.

Real-time speckle variance swept-source optical coherence tomography using a graphics processing unit.

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.