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.

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.

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.

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.

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.

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.

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.

Detecting apoptosis using dynamic light scattering with optical coherence tomography.

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.

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.

Oxygen-independent degradation of HIF-alpha via bioengineered VHL tumour suppressor complex.

Tumour hypoxia promotes the accumulation of the otherwise oxygen-labile hypoxia-inducible factor (HIF)-alpha subunit whose expression is associated with cancer progression, poor prognosis and resistance to conventional radiation and chemotherapy. The oxygen-dependent degradation of HIF-alpha is carried out by the von Hippel-Lindau (VHL) protein-containing E3 that directly binds and ubiquitylates HIF-alpha for subsequent proteasomal destruction. Thus, the cellular proteins involved in the VHL-HIF pathway have been recognized as attractive molecular targets for cancer therapy. However, the various compounds designed to inhibit HIF-alpha or HIF-downstream targets, although promising, have shown limited success in the clinic. In the present study, we describe the bioengineering of VHL protein that removes the oxygen constraint in the recognition of HIF-alpha while preserving its E3 enzymatic activity. Using speckle variance-optical coherence tomography (sv-OCT), we demonstrate the dramatic inhibition of angiogenesis and growth regression of human renal cell carcinoma xenografts upon adenovirus-mediated delivery of the bioengineered VHL protein in a dorsal skin-fold window chamber model. These findings introduce the concept and feasibility of 'bio-tailored' enzymes in the treatment of HIF-overexpressing tumours.

Drug and light dose responses to focal photodynamic therapy of single blood vessels in vivo.

As part of an ongoing program to develop two-photon (2-gamma) photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1-gamma and 2-gamma activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2-gamma cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2-gamma PDT of AMD.

Interstitial Doppler optical coherence tomography as a local tumor necrosis predictor in photodynamic therapy of prostatic carcinoma: an in vivo study.

We have tested the feasibility of real-time localized blood flow measurements, obtained with interstitial (IS) Doppler optical coherence tomography (DOCT), to predict photodynamic therapy (PDT)-induced tumor necrosis deep within solid Dunning rat prostate tumors. IS-DOCT was used to quantify the PDT-induced microvascular shutdown rate in s.c. Dunning prostate tumors (n=28). Photofrin (12.5 mg/kg) was administered 20 to 24 hours before tumor irradiation, with 635 nm surface irradiance of 8 to 133 mWcm(-2) for 25 minutes. High frequency ultrasound and calipers were used to measure the thickness of the skin covering the tumor and the location of the echogenic IS probe within it. A two-layer Monte Carlo model was used to calculate subsurface fluence rates within the IS-DOCT region of interest (ROI). Treatment efficacy was estimated by percent tumor necrosis within the ROI, as quantified by H&E staining, and correlated to the measured microvascular shutdown rate during PDT treatment. IS-DOCT measured significant PDT-induced vascular shutdown within the ROI in all tumors. A strong relationship (R2=0.723) exists between the percent tumor necrosis at 24 hours posttreatment and the vascular shutdown rate: slower shutdown corresponded to higher treatment efficacy, i.e., more necrosis. Controls (needle+light, no drug, n=3) showed minimal microvascular changes or necrosis (4%+/-1%). This study has correlated a biological end point with a direct and localized measurement of PDT-induced microvascular changes, suggesting a potential clinical role of on-line, real-time microvascular monitoring for optimizing treatment efficacy in individual patients.

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.

Feasibility of interstitial Doppler optical coherence tomography for in vivo detection of microvascular changes during photodynamic therapy.

INTRODUCTION: Doppler optical coherence tomography (DOCT) is an emerging imaging modality that provides subsurface microstructural and microvascular tissue images with near histological resolution and sub-mm/second velocity sensitivity. A key drawback of OCT for some applications is its shallow (1-3 mm) penetration depth. This fundamentally limits DOCT imaging to transparent, near-surface, intravascular, or intracavitary anatomical sites. Consequently, interstitial Doppler OCT (IS-DOCT) was developed for minimally-invasive in vivo imaging of microvasculature and microstructure at greater depths, providing access to deep-seated solid organs. Using Dunning prostate cancer in a rat xenograft model, this study evaluated the feasibility of IS-DOCT monitoring of microvascular changes deep within a tumor caused by photodynamic therapy (PDT). MATERIALS AND METHODS: The DOCT interstitial probe was constructed using a 22 G (diameter approximately 0.7 mm) needle, with an echogenic surface finish for enhanced ultrasound visualization. The lens of the probe consisted of a gradient-index fiber, fusion spliced to an angle-polished coreless tip to allow side-view scanning. The lens was then fusion spliced to a single-mode optical fiber that was attached to the linear scanner via catheters and driven along the longitudinal axis of the needle to produce a 2D subsurface DOCT image. The resultant IS-DOCT system was used to monitor microvascular changes deep within the tumor mass in response to PDT in the rat xenograft model of Dunning prostate cancer. Surface PDT was delivered at 635 nm with 40 mW of power, for a total light dose of 76 J/cm(2), using 12.5 mg/kg of Photofrin as the photosensitizer dose. RESULTS: IS-DOCT demonstrated its ability to detect microvasculature in vivo and record PDT-induced changes. A reduction of detected vascular cross sectional area during treatment and partial recovery post-treatment were observed. CONCLUSIONS: IS-DOCT is a potentially effective tool for real-time visualization and monitoring of the progress of PDT treatments. This capability may play an important role in elucidating the mechanisms of PDT in tumors, pre-treatment planning, feedback control for treatment optimization, determining treatment endpoints and post-treatment assessments.