Radiation-Activated Cobalamin-Kinase Inhibitors for Treatment of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most dismal diagnoses that a patient can receive. PDAC is extremely difficult to treat, as drug delivery is challenging in part due to the lack of vascularization, high stromal content, and high collagen content of these tumors. We have previously demonstrated that attaching drugs to the cobalamin scaffold provides selectivity for tumors over benign cells due to a high vitamin demand in these rapidly growing cells and an overexpression of transcobalamin receptors in a variety of cancer types. Importantly, we have shown the ability to deliver cobalamin derivatives to orthotopic pancreas tumors. Tyrosine kinase inhibitors have shown promise in treating PDAC as well as other cancer types. However, some of these inhibitors suffer from drug resistance, and as such, their success has been diminished. With this in mind, we synthesized the tyrosine kinase inhibitors erlotinib (EGFR) and dasatinib (Src) that are attached to this cobalamin platform. Both of these cobalamin-drug conjugates cause visible light-induced apoptosis, and the cobalamin-erlotinib conjugate (2) causes X-ray-induced apoptosis in MIA PaCa-2 cells. Both visible light and X-rays provide spatial control of drug release; however, utilizing X-ray irradiation offers the advantage of deeper tissue penetration. Therefore, we explored the utilization of 2 as a synergistic therapy with radiation in athymic nude mice implanted with MIA PaCa-2 tumors. We discovered that the addition of 2 caused an enhanced reduction in tumor margins in comparison with radiation therapy alone. In addition, treatment with 2 in the absence of radiation caused no significant reduction in tumor size in comparison with the controls. The cobalamin technology presented here allows for the spatial release of drugs in conjunction with external beam radiation therapy, potentially allowing for more effective treatment of deep-seated tumors with less systemic side effects.

Fluorescence molecular optomic signatures improve identification of tumors in head and neck specimens.

Background: Fluorescence molecular imaging using ABY-029, an epidermal growth factor receptor (EGFR)-targeted, synthetic Affibody peptide labeled with a near-infrared fluorophore, is under investigation for surgical guidance during head and neck squamous cell carcinoma (HNSCC) resection. However, tumor-to-normal tissue contrast is confounded by intrinsic physiological limitations of heterogeneous EGFR expression and non-specific agent uptake. Objective: In this preliminary study, radiomic analysis was applied to optical ABY-029 fluorescence image data for HNSCC tissue classification through an approach termed "optomics." Optomics was employed to improve tumor identification by leveraging textural pattern differences in EGFR expression conveyed by fluorescence. The study objective was to compare the performance of conventional fluorescence intensity thresholding and optomics for binary classification of malignant vs. non-malignant HNSCC tissues. Materials and Methods: Fluorescence image data collected through a Phase 0 clinical trial of ABY-029 involved a total of 20,073 sub-image patches (size of 1.8 × 1.8 mm2) extracted from 24 bread-loafed slices of HNSCC surgical resections originating from 12 patients who were stratified into three dose groups (30, 90, and 171 nanomoles). Each dose group was randomly partitioned on the specimen-level 75%/25% into training/testing sets, then all training and testing sets were aggregated. A total of 1,472 standardized radiomic features were extracted from each patch and evaluated by minimum redundancy maximum relevance feature selection, and 25 top-ranked features were used to train a support vector machine (SVM) classifier. Predictive performance of the SVM classifier was compared to fluorescence intensity thresholding for classifying testing set image patches with histologically confirmed malignancy status. Results: Optomics provided consistent improvement in prediction accuracy and false positive rate (FPR) and similar false negative rate (FNR) on all testing set slices, irrespective of dose, compared to fluorescence intensity thresholding (mean accuracies of 89% vs. 81%, P = 0.0072; mean FPRs of 12% vs. 21%, P = 0.0035; and mean FNRs of 13% vs. 17%, P = 0.35). Conclusions: Optomics outperformed conventional fluorescence intensity thresholding for tumor identification using sub-image patches as the unit of analysis. Optomics mitigate diagnostic uncertainties introduced through physiological variability, imaging agent dose, and inter-specimen biases of fluorescence molecular imaging by probing textural image information. This preliminary study provides a proof-of-concept that applying radiomics to fluorescence molecular imaging data offers a promising image analysis technique for cancer detection in fluorescence-guided surgery.

Improved Discrimination of Tumors with Low and Heterogeneous EGFR Expression in Fluorescence-Guided Surgery Through Paired-Agent Protocols.

PURPOSE: The goal of fluorescence-guided surgery (FGS) in oncology is to improve the surgical therapeutic index by enhancing contrast between cancerous and healthy tissues. However, optimal discrimination between these tissues is complicated by the nonspecific uptake and retention of molecular targeted agents and the variance of fluorescence signal. Paired-agent imaging (PAI) employs co-administration of an untargeted imaging agent with a molecular targeted agent, providing a normalization factor to minimize nonspecific and varied signals. The resulting measured binding potential is quantitative and equivalent to in vivo immunohistochemistry of the target protein. This study demonstrates that PAI improves the accuracy of tumor-to-healthy tissue discrimination compared to single-agent imaging for in vivo FGS. PROCEDURES: PAI using a fluorescent anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) with untargeted IRDye 700DX carboxylate was compared to ABY-029 alone in an oral squamous cell carcinoma xenograft mouse model at 3 h after dye administration (n = 30). RESULTS: PAI significantly enhanced tumor discrimination, as compared to ABY-029 alone in low EGFR-expressing tumors and highly heterogeneous populations including multiple cell lines with varying expression (diagnostic accuracy: 0.908 vs. 0.854 and 0.908 vs. 0.822; and ROC curve AUC: 0.963 vs. 0.909 and 0.957 vs. 0.909, respectively) indicating a potential for universal FGS image thresholds to determine surgical margins. In addition, PAI achieved significantly higher diagnostic ability than ABY-029 alone 0.25-5-h post injection and exhibited a stronger correlation to EGFR expression heterogeneity. CONCLUSION: The quantitative receptor delineation of PAI promises to improve the surgical therapeutic index of cancer resection in a clinically relevant timeline.

High optical-throughput spectroscopic singlet oxygen and photosensitizer luminescence dosimeter for monitoring of photodynamic therapy.

We report a high light-throughput spectroscopic dosimeter system that is able to noninvasively measure luminescence signals of singlet oxygen (1 O2 ) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7 nm). This is adequate to detect 1 O2 phosphorescence in the presence of strong luminescence background in vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of 1 O2 phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with 1 O2 phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time 1 O2 dosimetry during PDT treatment using CW light.

Quantification of Oxygen Depletion During FLASH Irradiation In Vitro and In Vivo.

PURPOSE: Delivery of radiation at ultrahigh dose rates (UHDRs), known as FLASH, has recently been shown to preferentially spare normal tissues from radiation damage compared with tumor tissues. However, the underlying mechanism of this phenomenon remains unknown, with one of the most widely considered hypotheses being that the effect is related to substantial oxygen depletion upon FLASH, thereby altering the radiochemical damage during irradiation, leading to different radiation responses of normal and tumor cells. Testing of this hypothesis would be advanced by direct measurement of tissue oxygen in vivo during and after FLASH irradiation. METHODS AND MATERIALS: Oxygen measurements were performed in vitro and in vivo using the phosphorescence quenching method and a water-soluble molecular probe Oxyphor 2P. The changes in oxygen per unit dose (G-values) were quantified in response to irradiation by 10 MeV electron beam at either UHDR reaching 300 Gy/s or conventional radiation therapy dose rates of 0.1 Gy/s. RESULTS: In vitro experiments with 5% bovine serum albumin solutions at 23°C resulted in G-values for oxygen consumption of 0.19 to 0.21 mm Hg/Gy (0.34-0.37 µM/Gy) for conventional irradiation and 0.16 to 0.17 mm Hg/Gy (0.28-0.30 µM/Gy) for UHDR irradiation. In vivo, the total decrease in oxygen after a single fraction of 20 Gy FLASH irradiation was 2.3 ± 0.3 mm Hg in normal tissue and 1.0 ± 0.2 mm Hg in tumor tissue (P < .00001), whereas no decrease in oxygen was observed from a single fraction of 20 Gy applied in conventional mode. CONCLUSIONS: Our observations suggest that oxygen depletion to radiologically relevant levels of hypoxia is unlikely to occur in bulk tissue under FLASH irradiation. For the same dose, FLASH irradiation induces less oxygen consumption than conventional irradiation in vitro, which may be related to the FLASH sparing effect. However, the difference in oxygen depletion between FLASH and conventional irradiation could not be quantified in vivo because measurements of oxygen depletion under conventional irradiation are hampered by resupply of oxygen from the blood.

Visualization and quantification of pancreatic tumor stroma in fresh tissue via ultraviolet surface excitation.

SIGNIFICANCE: The study has confirmed the feasibility of using ultraviolet (UV) excitation to visualize and quantify desmoplasia in fresh tumor tissue of pancreatic adenocarcinoma (PDAC) in an orthotopic xenograft mouse model, which provides a useful imaging platform to evaluate acute therapeutic responses. AIM: Stromal network of collagen prominent in PDAC tumors is examined by imaging fresh tissue samples stained with histological dyes. Fluorescence signals are color-transferred to mimic Masson's trichrome staining. APPROACH: Murine tumor samples were stained with Hoechst, eosin, and rhodamine B and excited at 275-nm. Fluorescence signals in the visible spectrum were captured by a CMOS color camera with high contrast and resolution at whole-tumor slice field of view. RESULTS: Fluorescence imaging using UV excitation is capable of visualizing collagen deposition in PDAC tumors. Both fluorescence and histology data showed collagen content of up to 30%. The collagen modulation effect due to photodynamic priming treatment was observed showing 13% of collagen reduction. Necrosis area is visible and perfusion imaging using Texas Red dextran is feasible. CONCLUSIONS: The study demonstrates collagen visualization in fresh PDAC tumor samples using UV excitation. This imaging platform also provides quantitative stromal information from fiber analysis and visibility of necrosis and perfusion, suitable for therapeutic response assessment of photodynamic therapy.

Evaluation of bone perfusion during open orthopedic surgery using quantitative dynamic contrast-enhanced fluorescence imaging.

In this study, an indocyanine green (ICG)-based dynamic contrast- enhanced fluorescence imaging (DCE-FI) technique was evaluated as a method to provide objective real-time data on bone perfusion using a porcine osteotomy model. DCE-FI with sequentially increasing injury to osseous blood supply was performed in 12 porcine tibias. There were measurable, reproducible and predictable changes to DCE-FI data across each condition have been observed on simple kinetic curve-derived variables as well variables derived from a novel bone-specific kinetic model. The best accuracy, sensitivity and specificity of 89%, 88% and 90%, have been achieved to effectively differentiate injured from normal/healthy bone.

Single pixel hyperspectral Cherenkov-excited fluorescence imaging with LINAC X-ray sheet scanning and spectral unmixing.

Cherenkov light induced from megavolt (MV) X-rays during external beam radiotherapy serves as an internal light source to excite phosphors or fluorophores within biological tissues for molecular imaging. The broad spectrum of Cherenkov light leads to significant spectral overlap with any luminescence emission and, to overcome this problem, a single pixel hyperspectral imaging methodology was demonstrated here by coupling the detection with light sheet scanning and filtered back projection reconstruction of hyperspectral images. Thin scanned sheets of MV X-rays produce Cherenkov light to illuminate the planes deep within the tissue-simulating media. A fluorescence probe was excited by Cherenkov light, and a complete hyperspectral sinogram of the data was obtained through translation and rotation of the beam. Hyperspectral 2D images finally were reconstructed. Through this approach of spectral unmixing, it was possible to resolve hyperspectral images of both the Cherenkov and resulting fluorescence intensity from molecular sensors.

Implantable sensor for local Cherenkov-excited luminescence imaging of tumor pO2 during radiotherapy.

SIGNIFICANCE: The necessity to use exogenous probes for optical oxygen measurements in radiotherapy poses challenges for clinical applications. Options for implantable probe biotechnology need to be improved to alleviate toxicity concerns in human use and facilitate translation to clinical trial use. AIM: To develop an implantable oxygen sensor containing a phosphorescent oxygen probe such that the overall administered dose of the probe would be below the Federal Drug Administration (FDA)-prescribed microdose level, and the sensor would provide local high-intensity signal for longitudinal measurements of tissue pO2. APPROACH: PtG4, an oxygen quenched dendritic molecule, was mixed into an agarose matrix at 100 µM concentration, allowing for local injection into tumors at the total dose of 10 nmol per animal, forming a gel at the site of injection. Cherenkov-excited luminescence imaging (CELI) was used to acquire the phosphorescence and provide intratumoral pO2. RESULTS: Although PtG4 does not form covalent bonds with agarose and gradually leaches out into the surrounding tissue, its retention time within the gel was sufficiently long to demonstrate the capability to measure intratumoral pO2 with the implantable gel sensors. The sensor's performance was first evaluated in vitro in tissue simulation phantoms, and then the sensor was used to measure changes in oxygen in MDA-MB-231 tumors during hypofractionated radiotherapy. CONCLUSIONS: Our study demonstrates that implantable oxygen sensors in combination with CELI present a promising approach for quantifying oxygen changes during the course of radiation therapy and thus for evaluating the tumor response to radiation. By improving the design of the gel-probe composition in order to prevent leaching of the probe into the tissue, biosensors can be created that should allow longitudinal oxygen measurements in tumors by means of CELI while using FDA-compliant microdose levels of the probe and thus lowering toxicity concerns.

X-ray-Induced Cherenkov Optical Triggering of Caged Doxorubicin Released to the Nucleus for Chemoradiation Activation.

Concurrent chemoradiotherapy is used for advanced cancers, but the chemotherapy is dose limited by normal tissue toxicity. Localized X-ray activation of chemotherapy could overcome this, as studied here, with release from self-assembled nanomicelles (NMs) created from copolymers loaded with doxorubicin (DOX) having a photocleavable o-nitrobenzyl ester (o-Ne) group. The micelles demonstrated release of DOX from X-ray-induced Cherenkov light and conversion from a caged hydrophobic form to hydrophilic DOX, which achieves nuclear localization. Folate on the exterior of the NMs directed them for effective intracellular uptake prior to irradiation. Irradiation with 8 Gy released the DOX, which then entered the cell nucleus, providing near-complete in vivo tumor eradication and negligible off-target organ damage. Micelles were assembled from molecular component materials that are commonly in human use. This study realizes triple targeting in chemoradiation with potential for cell-receptor-mediated uptake, localized radiotherapy activation, and nuclear relocalization, all leading to limited off-target toxicity.

Dual-agent fluorescent labeling of soft-tissue sarcomas improves the contrast based upon targeting both interstitial and cellular components of the tumor milieu.

BACKGROUND: Current practices for fluorescence-guided cancer surgery utilize a single fluorescent agent, but homogeneous distribution throughout the tumor is difficult to achieve. We hypothesize that administering a perfusion and a molecular-targeted agent at their optimal administration-to-imaging time will improve whole-tumor contrast. EXPERIMENTAL DESIGN: Mice bearing subcutaneous xenograft human synovial sarcomas were administered indocyanine green (ICG) (3 mg/kg) or ABY-029 (48.7 µg/kg)-an epidermal growth factor receptor-targeted Affibody molecule-alone or in combination. Fluorescence contrast and signal distribution were compared between treatment groups. Two commercial fluorescence imaging systems were tested for simultaneous imaging of ICG and ABY-029. RESULTS: ABY-029 has a moderate positive correlation with viable tumor (ρ = 0.2 ± 0.4), while ICG demonstrated a strong negative correlation (ρ = -0.6 ± 0.1). The contrast-to-variance ratio was highest in the ABY-029 +ICG (2.5 ± 0.8), compared to animals that received ABY-029 (2.3 ± 0.8) or ICG (2.0 ± 0.5) alone. Moreover, the combination of ABY-029 + ICG minimizes the correlation between viable tumor and fluorescence intensity (ρ = -0.1 ± 0.2) indicating the fluorescence signal distribution is more homogeneous throughout the tumor milieu. CONCLUSION: Dual-agent imaging utilizing a single channel in a commercial fluorescence-guided imaging system tailored for IRDye 800CW is a promising method to increase tumor contrast in a clinical setting.

Endosteal and periosteal blood flow quantified with dynamic contrast-enhanced fluorescence to guide open orthopaedic surgery.

Due to the lack of objectively measurable or quantifiable methods to assess the bone perfusion, the success of removing devitalized bone is based almost entirely on surgeon's experience and varies widely across surgeons and centers. In this study, an indocyanine green (ICG)-based dynamic contrast-enhanced fluorescence imaging (DCE-FI) has been developed to objectively assess bone perfusion and guide surgical debridement. A porcine trauma model (n = 6 pigs × 2 legs) with up to 5 conditions of severity in loss of flow in each, was imaged by a commercial fluorescence imaging system. By applying the bone-specific hybrid plug-compartment (HyPC) kinetic model to four-minute video sequences, the perfusion-related metrics, such as peak intensity, total bone blood flow (TBBF) and endosteal bone blood flow to TBBF fraction (EFF) were calculated. The results shown that the combination of TBBF and EFF can effectively differentiate injured from normal bone with the accuracy, sensitivity and specificity of 89%, 88% and 90%, respectively. Our subsequent first in human bone blood flow imaging study confirmed DCE-FI can be successfully translated into human orthopaedic trauma patients.

Imaging of hypoxia, oxygen consumption and recovery in vivo during ALA-photodynamic therapy using delayed fluorescence of Protoporphyrin IX.

BACKGROUND: Hypoxic lesions often respond poorly to cancer therapies. Particularly, photodynamic therapy (PDT) consumes oxygen in treated tissues, which in turn lowers its efficacy. Tools for online monitoring of intracellular pO2 are desirable. METHODS: The pO2 changes were tracked during photodynamic therapy (PDT) with δ-aminolevulinic acid (ALA) in mouse skin, xenograft tumors, and human skin. ALA was applied either topically as Ameluz cream or systemically by injection. Mitochondrial pO2 was quantified by time-gated lifetime-based imaging of delayed fluorescence (DF) of protoporphyrin IX (PpIX). RESULTS: pO2-weighted images were obtained with capture-times of several seconds, radiant exposures near 10 mJ/cm2, spatial resolution of 0.3 mm, and a broad dynamic range 1-50 mmHg, corresponding to DF lifetimes ≈20-2000 µs. The dose-rate effect on oxygen consumption was investigated in mouse skin. A fluence rate of 1.2 mW/cm2 did not cause any appreciable oxygen depletion, whereas 6 mW/cm2 and 12 mW/cm2 caused severe oxygen depletion after radiant exposures of only 0.4-0.8 J/cm2 and <0.2 J/cm2, respectively. Reoxygenation after PDT was studied too. With a 5 J/cm2 radiant exposure, the recovery times were 10-60 min, whereas with 2 J/cm2 they were only 1-6 min. pO2 distribution was spatially non-uniform at (sub)-millimeter scale, which underlines the necessity of tracking pO2 changes by imaging rather than point-detection. CONCLUSIONS: Time-gated imaging of PpIX DF seems to be a unique tool for direct online monitoring of pO2 changes during PDT with a promising potential for research purposes as well as for comparatively easy clinical translation to improve efficacy in PDT treatment.

Tracking tumor radiotherapy response in vivo with Cherenkov-excited luminescence ink imaging.

This study demonstrates remote imaging for in vivo detection of radiation-induced tumor microstructural changes by tracking the diffusive spread of injected intratumor UV excited tattoo ink using Cherenkov-excited luminescence imaging (CELI). Micro-liter quantities of luminescent tattoo ink with UV absorption and visible emission were injected at a depth of 2 mm into mouse tumors prior to receiving a high dose treatment of radiation. X-rays from a clinical linear accelerator were used to excite phosphorescent compounds within the tattoo ink through Cherenkov emission. The in vivo phosphorescence was detected using a time-gated intensified CMOS camera immediately after injection, and then again at varying time points after the ink had broken down with the apoptotic tumor cells. Ex vivo tumors were imaged post-mortem using hyperspectral cryo-fluorescence imaging to quantify necrosis and compared to Cherenkov-excited light imaging of diffusive ink spread measured in vivo. Imaging of untreated control mice showed that ink distributions remained constant after four days with less than 3% diffusive spread measured using full width at 20% max. For all mice, in vivo CELI measurements matched within 12% of the values estimated by the high-resolution ex vivo sliced luminescence imaging of the tumors. The tattoo ink spread in treated mice was found to correlate well with the nonperfusion necrotic core volume (R2 = 0.92) but not well with total tumor volume changes (R2 = 0.34). In vivo and ex vivo findings indicate that the diffusive spread of the injected tattoo ink can be related to radiation-induced necrosis, independent of total tumor volume change. Tracking the diffusive spread of the ink allows for distinguishing between an increase in tumor size due to new cellular growth and an increase in tumor size due to edema. Furthermore, the imaging resolution of CELI allows for in vivo tracking of subtle microenvironmental changes which occur earlier than tumor shrinkage and this offers the potential for novel, minimally invasive radiotherapy response assay without interrupting a singular clinical workflow.

Multispectral singlet oxygen and photosensitizer luminescence dosimeter for continuous photodynamic therapy dose assessment during treatment.

SIGNIFICANCE: Photodynamic therapy (PDT) involves complex light-drug-pathophysiology interactions that can be affected by multiple parameters and often leads to large variations in treatment outcome from patient to patient. Direct PDT dosimetry technologies have been sought to optimize the control variables (e.g., light dose, drug administration, tissue oxygenation, and patient conditioning) for best patient outcomes. In comparison, singlet oxygen (O21) dosimetry has been tested in various forms to provide an accurate and perhaps comprehensive prediction of the treatment efficacy. AIM: We discuss an advanced version of this approach provided by a noninvasive, continuous wave dosimeter that can measure near-infrared spectrally resolved luminescence of both photosensitizer (PS) and O21 generated during PDT cancer treatment. APPROACH: This dosimetry technology uses an amplified, high quantum efficiency InGaAs detector with spectroscopic decomposition during the light treatment to continuously extract the maximum signal of O21 phosphorescence while suppressing the strong PS luminescence background by spectrally fitting the data points across nine narrow band wavelengths. O21 and PS luminescence signals were measured in vivo in FaDu xenograft tumors grown in mice during PDT treatment using Verteporfin as the PS and a continuous laser treatment at 690 nm wavelength. RESULTS: A cohort of 19 mice was used and observations indicate that the tumor growth rate inhibition showed a stronger correlation with O21 than with just the PS signal. CONCLUSIONS: These results suggest that O21 measurement may be a more direct dosimeter of PDT damage, and it has potential value as a definitive diagnostic for PDT treatment, especially with spectral separation of the background luminescence and online estimation of the PS concentration.

Imaging luminescent tattoo inks for direct visualization of linac and cobalt irradiation.

PURPOSE: Tattoo fiducials are commonly used in radiotherapy patient alignment, and recent studies have examined the use of UV-excited luminescent tattoo ink as a cosmetic substitute to make these visible under UV illumination. The goal of this study was to show how luminescent tattoo inks could be excited with MV radiation and imaged during beam delivery for direct visualization of field position. METHODS: A survey of nine UV-sensitive tattoo inks with various emission spectra were investigated using both UV and MV excitation. Images of liquid solutions were collected under MV excitation using an intensified-CMOS imager. Solid skin-simulating phantoms were imaged with both surface-painted ink and in situ tattooing during dose delivery by both a clinical linear accelerator and cobalt-60 source. RESULTS: The UV inks have peak fluorescence emission ranging from approximately 440 to 600 nm with lifetimes near 11-16 µs. The luminescence intensity is approximately 6x higher during the x-ray pulse than after the pulse, however, the signal-to-noise is only approximately twice as large. Spatial resolution for imaging was achieved at 1.6 mm accuracy in a skin test phantom. Optical filtering allows for continuous imaging using a cobalt source and provides a mechanism to discriminate ink colors using a monochromatic image sensor. CONCLUSIONS: This study demonstrates how low-cost inks can be used as fiducial markers and imaged both using time-gated and continuous modes during MV dose delivery. Phantom studies demonstrate the potential application of real-time field verification. Further studies are required to understand if this technique could be used as a tool for radiation dosimetry.

Imaging of singlet oxygen feedback delayed fluorescence and lysosome permeabilization in tumor in vivo during photodynamic therapy with aluminum phthalocyanine.

Significance: Singlet oxygen is a key cytotoxic agent in photodynamic therapy (PDT). As such, its imaging is highly desirable, but existing direct imaging methods are still limited by the exceptionally low yield of the luminescence signal. Singlet oxygen feedback delayed fluorescence (SOFDF) of the photosensitizer is a higher yield alternative for indirect measurement of this signal.

Aim: The aim was to explore feasibility of SOFDF imaging in vivo in tumor-bearing mice during PDT and investigate how SOFDF images can be transformed into images of singlet oxygen. In addition, we study whether lysosome permeabilization can be visualized through fluorescence lifetime.

Approach: Mice were intravenously injected with 2.5 mg/kg of photosensitizer aluminum(III) phthalocyanine tetrasulfonate (AlPcS4) 20 h prior to experiments, having subcutaneous BxPC3 pancreas tumors. Time-resolved delayed fluorescence and prompt fluorescence (PF) were imaged using an intensified time-gated camera with 10-Hz pulsed laser excitation at 690 nm.

Results: Delayed emission from AlPcS4 was detected with lifetimes 7 to 11 µs, which was attributed to SOFDF and shown to be oxygen-dependent. Singlet oxygen images were approximated by the ratio of SOFDF/PF at each pixel. SOFDF images of a good quality could be captured within several seconds with a radiant exposure of ∼20 mJ / cm2. In addition, lifetime images of AlPcS4 PF in ns-time domain enabled us to visualize the event of lysosome permeabilization, as the lifetime increased from ∼4.7 to 5.2 ns.

Conclusions: Imaging of SOFDF in vivo in mouse tumor during PDT with AlPcS4 is feasible, and it is a promising method for singlet molecular oxygen monitoring. Moreover, the time-gated approach also enables visualization of the lysosome permeabilization that alters the PF lifetime.

High-Resolution Ex Vivo Elastography to Characterize Tumor Stromal Heterogeneity In Situ in Pancreatic Adenocarcinoma.

OBJECTIVE: Tumor stiffening in pancreatic adenocarcinoma (PDAC) has been linked to cancer progression and lack of therapy response, yet current elastography tools cannot map stiffness in a whole tumor field-of-view with biologically relevant spatial resolution. Therefore, this study was developed to assess stiffness heterogeneity and geometrical patterns across whole PDAC xenograft ex vivo tumors. METHODS: The ex vivo elastography (EVE) mapping system was capable of creating stiffness map at 300-micron spatial resolution under a 5-20 mm field of view relevant to whole tumor assessment. The stiffness value at each location was determined by compression testing and an absolute tumor Young's modulus map was calculated based on the calibration between the system and ultrasound elastography (R2 = 0.95). RESULTS: Two PDAC tumor lines AsPC-1 and BxPC-3 implanted in xenograft models were assessed to show tumor stiffness and its linear relationship to collagen content (R2 = 0.59). EVE was able to capture stiffness heterogeneity ranging between 5 and 100 kPa in pancreatic tumors with collagen content up to 25%. More importantly, data shows the inverse relationship of local stiffness to local drug distribution (R2 = 0.66) and vessel patency (R2 = 0.61) in both PDAC tumor lines. CONCLUSION: The results suggested that elastography could be utilized to predict drug penetration in PDAC tumors or assess response to biological modifying adjunct therapies. SIGNIFICANCE: This study presents the first attempt to map out stiffness on a biologically relevant spatial scale across whole PDAC tumor slices with spatial resolution in the hundreds of microns.

Tissue pO2 distributions in xenograft tumors dynamically imaged by Cherenkov-excited phosphorescence during fractionated radiation therapy.

Hypoxia in solid tumors is thought to be an important factor in resistance to therapy, but the extreme microscopic heterogeneity of the partial pressures of oxygen (pO2) between the capillaries makes it difficult to characterize the scope of this phenomenon without invasive sampling of oxygen distributions throughout the tissue. Here we develop a non-invasive method to track spatial oxygen distributions in tumors during fractionated radiotherapy, using oxygen-dependent quenching of phosphorescence, oxygen probe Oxyphor PtG4 and the radiotherapy-induced Cherenkov light to excite and image the phosphorescence lifetimes within the tissue. Mice bearing MDA-MB-231 breast cancer and FaDu head neck cancer xenografts show different pO2 responses during each of the 5 fractions (5 Gy per fraction), delivered from a clinical linear accelerator. This study demonstrates subsurface in vivo mapping of tumor pO2 distributions with submillimeter spatial resolution, thus providing a methodology to track response of tumors to fractionated radiotherapy.

Tumor targeting vitamin B12 derivatives for X-ray induced treatment of pancreatic adenocarcinoma.

BACKGROUND: X-Ray induced phototherapy is highly sought after as it provides a deep tissue, synergistic method of treating cancers via standard-of-care radiotherapy. When this is combined with releasable chemotherapy agents, it can provide high target selectivity, with reduced off-target organ effects that limit current systemic therapies. We have recently developed a unique light-activated drug delivery system whereby the drug is conjugated to an alkylcobalamin scaffold. Alkylcobalamins are actively transported into cells by transcobalamin receptors (TCblR), which are overexpressed in a variety of cancer types. We hope to utilize this cobalamin scaffold technology for drug delivery in pancreatic adenocarcinoma (PDAC) cancer. METHODS: The ability of the cobalamin scaffold to selectively target PDAC was investigated by treating mice that had MIA PaCa-2 xenografts with an alkylcobalamin labeled with the fluorophore Bodipy650 (Bodipy650-cobalamin). The mice were imaged alive and organs as well as tumors were subsequently imaged ex vivo. In addition, we examined the potential of the cobalamin scaffold to deliver drugs to orthotopic pancreas MIA PaCa-2 tumors with Bodipy650-cobalamin. We determined the light dose required for release of cargo from the cobalamin scaffold by examining the fluorescence increase of Bodipy650-cobalamin in response to red light (650 nm). Finally, we probed the ability of the cobalamin scaffold to release cargo with increasing X-ray doses from a clinical linear accelerator. RESULTS: We have found that Bodipy650-cobalamin was shown to localize in MIA PaCa-2 tumors, both in flank and orthotopic models. We quantified a light dose for red light release from the cobalamin scaffold that is within normal clinical doses required for photodynamic therapy. This derivative was also activated with clinical X-ray doses from a linear accelerator. CONCLUSIONS: Tumor selectivity combined with fluorescence detection demonstrates the effectiveness of the vitamin B12 scaffold as a theranostic targeting agent. The activation of this scaffold with radiation from a linear accelerator shows potential for action as radiation-induced chemotherapy.