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Radiation damage studies are used to optimize radiotherapy treatment techniques. Although biological indicators of damage are the best assays of effect, they are highly variable due to biological heterogeneity. The free radical radiochemistry can be assayed with optical reporters, allowing for high precision titration of techniques. We examine the optical reporters of radiochemistry to highlight those with the best potential for translational use , as surrogates for biological damage assays, to inform on mechanisms. A survey of the radical chemistry effects from reactive oxygen species (ROS) and oxygen itself was completed to link to DNA or biological damage. Optical reporters of ROS include fluorescent, phosphorescent, and bioluminescent molecules that have a variety of activation pathways, and each was reviewed for its translation potential. There are molecular reporters of ROS having potential to report within living systems, including derivatives of luminol, 2'7'-dichlorofluorescein diacetate, Amplex Red, and fluorescein. None have unique specificity to singular ROS species. Macromolecular engineered reporters unique to specific ROS are emerging. The ability to directly measure oxygen via reporters, such as Oxyphor and protoporphyrin IX, is an opportunity to quantify the consumption of oxygen during ROS generation, and this translates from to use. Emerging techniques, such as ion particle beams, spatial fractionation, and ultra-high dose rate FLASH radiotherapy, provide the motivation for these studies. optical reporters of radiochemistry are quantitatively useful for comparing radiotherapy techniques, although their use comes at the cost of the unknown connection to the mechanisms of radiobiological damage. Still their lower measurement uncertainty, compared with biological response assay, makes them an invaluable tool. Linkage to DNA damage and biological damage is needed, and measures such as oxygen consumption serve as useful surrogate measures that translate to use.

The development of fluorescent contrast agents for fluorescence-guided surgery is rapidly growing with many agents being designed for tumor visualization. Although efforts have been made to standardize the sensitivity of imaging system detection methods for these contrast agents, guidelines to evaluate tumor contrast agent performance, especially the selection of tumor and background regions of interest (ROIs), differ widely across studies. We examine how systematically changing tumor and background ROIs affects common metrics of contrast agent performance. We aim to elucidate the influence of changing tumor and background brain regions of interest on fluorescent contrast agent performance. Mice with orthotopic brain tumors were administered a non-targeted fluorescent contrast agent 40 min prior to sacrifice and then imaging of the specimen using whole-body fluorescence cryotomography. The reconstructed 3D fluorescence volumes were then used to compute contrast and diagnostic performance metrics [tumor-to-background brain ratio (TBR), contrast-to-noise (CNR), and area under the receiver operating characteristic curve (AUC)] while systematically varying tumor and normal brain ROIs. ROI selection had a significant impact on the reported values of metrics used to evaluate fluorescence imaging strategies. The use of contralateral background ROIs, commonly used in the field, produced elevated and favorable performance metric values. These metrics decreased as background ROIs approached regions adjacent to the tumor boundary. TBR changed by a factor of 5, CNR by a factor of 7, and AUC by over 10%, largely depending on the proximity of the background region to the tumor. Background ROI selection has a significant impact on the performance metrics commonly used in the field. Future studies should carefully select ROIs relevant to the application and include clear descriptions of these regions.

ALA-PpIX and second-window indocyanine green (ICG) have been studied widely for guiding the resection of high-grade gliomas. These agents have different mechanisms of action and uptake characteristics, which can affect their performance as surgical guidance agents. Elucidating these differences in animal models that approach the size and anatomy of the human brain would help guide the use of these agents. Herein, we report on the use of a new pig glioma model and fluorescence cryotomography to evaluate the 3D distributions of both agents throughout the whole brain. We aim to assess and compare the 3D spatial distributions of ALA-PpIX and second-window ICG in a glioma-bearing pig brain using fluorescence cryotomography. A glioma was induced in the brain of a transgenic Oncopig via adeno-associated virus delivery of Cre-recombinase plasmids. After tumor induction, the pro-drug 5-ALA and ICG were administered to the animal 3 and 24 h prior to brain harvest, respectively. The harvested brain was imaged using fluorescence cryotomography. The fluorescence distributions of both agents were evaluated in 3D in the whole brain using various spatial distribution and contrast performance metrics. Significant differences in the spatial distributions of both agents were observed. Indocyanine green accumulated within the tumor core, whereas ALA-PpIX appeared more toward the tumor periphery. Both ALA-PpIX and second-window ICG provided elevated tumor-to-background contrast (13 and 23, respectively). This study is the first to demonstrate the use of a new glioma model and large-specimen fluorescence cryotomography to evaluate and compare imaging agent distribution at high resolution in 3D.

Tissue oxygenation is well understood to impact radiosensitivity, with reports demonstrating a significant effect of breathing condition and anesthesia type on tissue oxygenation levels and radiobiological response. However, the temporal kinetics of intracellular and extracellular oxygenation have never been quantified, on the timescale that may affect radiotherapy studies. C57BL/6 mice were anesthetized using isoflurane at various percentages or ketamine/xylazine (ket/xyl: 100/10 mg/kg) ( N  = 48). Skin pO 2 was measured using Oxyphor PdG4 and tracked after anesthetization began. Oxyphor data was validated with relative measurements of intracellular oxygen via protoporphyrin IX (PpIX) delayed fluorescence (DF) imaging. Ex vivo localization of both PdG4 Oxyphor and PpIX were quantified. Under all isoflurane anesthesia conditions, leg skin pO 2 levels significantly increased from 12 to 15 mmHg at the start of anesthesia induction (4–6 min) to 24–27 mmHg after 10 min ( p  < 0.05). Ketamine/xylazine anesthesia led to skin pO 2 maintained at 15–16 mmHg throughout the 10-minute study period ( p  < 0.01). An increase of pO 2 in mice breathing isoflurane was demonstrated with Oxyphor and PpIX DF, indicating similar intracellular and extracellular oxygenation. These findings demonstrate the importance of routine anesthesia administration, where consistency in the timing between induction and irradiation may be crucial to minimizing variability in radiation response.

Purpose ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. Procedures Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 μg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. Results Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. Conclusion Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5 mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

Realizing the full potential of iron oxide nanoparticles (IONP) for cancer diagnosis and therapy requires selective tumor cell accumulation. Here, we report a systematic analysis of two key determinants for IONP homing to human breast cancers: (i) particle size and (ii) active vs passive targeting. In vitro, molecular targeting to the HER2 receptor was the dominant factor driving cancer cell association. In contrast, size was found to be the key determinant of tumor accumulation in vivo, where molecular targeting increased tumor tissue concentrations for 30 nm but not 100 nm IONP. Similar to the in vitro results, PEGylation did not influence in vivo IONP biodistribution. Thus, the results reported here indicate that the in vitro advantages of molecular targeting may not consistently extend to pre-clinical in vivo settings. These observations may have important implications for the design and clinical translation of advanced, multifunctional, IONP platforms.

Fluorescence imaging has the potential to significantly improve neurosurgical resection of oncologic lesions through improved differentiation between normal and cancerous tissue at the tumor margins. In order to successfully mark glioma tissue a fluorescent tracer must have the ability to penetrate through the blood brain barrier (BBB) and provide delineation in the tumor periphery where heterogeneously intact BBB may exist. In this study it was hypothesized that, due to its smaller size, fluorescently labeled anti-EGFR Affibody protein (∼7 kDa) would provide a more clear delineation of the tumor margin than would fluorescently labeled cetuximab, a full antibody (∼150 kDa) to the epidermal growth factor receptor (EGFR). Cetuximab and anti-EGFR targeted Affibody were conjugated to two different fluorescent dyes (both emitting in the near-infrared) and injected intravenously into 6 athymic mice which were inoculated orthotopically with green fluorescent protein (GFP) expressing human U251 glioma cells. Each mouse was sacrificed at 1-h post injection, at which time brains were removed, snap frozen, sectioned and quantitatively analyzed for fluorescence distribution. Ex vivo analysis showed on average, nearly equal concentrations of cetuximab and Affibody within the tumor (on average Affibody made up 49±6% of injected protein), however, the cetuximab was more confined to the center of the tumor with Affibody showing significantly higher concentrations at the tumor periphery (on average Affibody made up 72±15% of injected protein in the outer 50 um of the tumor). Further ex vivo analysis of detection studies showed that the Affibody provided superior discrimination for differentiation of tumor from surrounding normal brain. The present study indicates that fluorescently labeled anti-EGFR Affibody can provide significantly better delineation of tumor margins than a fluorescently labeled anti-EGFR antibody and shows considerable potential for guiding margin detection during neurosurgery.

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class 1 . Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells 2 , 3 , 4 , 5 , 6 . Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.

Background Hyperthermia is one of the promising cancer treatment strategies enabled by local heating with the use of tumor-targeting magnetic nanoparticles (MNP) under a non-invasive magnetic field. However, one of the remaining challenges is how to achieve therapeutic levels of heat (without causing damages to regular tissues) in tumors that cannot be effectively treated with anti-tumor drug delivery. Results In this work, we report a facile method to fabricate magnetic nanorods for hyperthermia by one-step wet chemistry synthesis using 3-Aminopropyltrimethoxysilane (APTMS) as the shape-controlling agent and ferric and ferrous ions as precursors. By adjusting the concentration of APTMS, hydrothermal reaction time, ratios of ferric to ferrous ions, magnetic nanorods with aspect ratios ranging from 4.4 to 7.6 have been produced. At the clinically recommended field strength of 300 Oe (or less) and the frequency of 184 kHz, the specific absorption rate (SAR) of these nanorods is approximately 50 % higher than that of commercial Bionized NanoFerrite particles. Conclusions This increase in SAR, especially at low field strengths, is crucial for treating deep tumors, such as pancreatic and rectal cancers, by avoiding the generation of harmful eddy current heating in normal tissues.

An improved method to estimate dose to esophageal tissue was investigated in the setting of photodynamic therapy with aminolevulinic acid‐induced protoporphyrin IX (PpIX) treatment. A model of treatment‐induced edema in the esophagus mucosa proved to be a well controlled and useful way to test the dosimetry model, and the light from the treatment laser together with the PpIX fluorescence intensity could be quantified reliably in real time. Dosimetry calculations based upon the detected fluorescence and bleaching kinetics were used to calculate the “effective” dose to the tissue, and a correlation was shown to exist between this metric and the edema induced in the esophagus. The difference between animals with no detectable treatment effect and those with significant edema was predictable based upon the dose calculation. The underlying assumption in the interpretation of the data is that rapid photobleaching of PpIX occurs when there is ample oxygen supply, and this bleaching is not present when oxygen is limited. This leads to the prediction that integration of the light and drug dose, in intervals where appreciable photobleaching occurs, should provide a prediction of the relative dose of singlet oxygen produced. This detection system and rodent model can be used for prospective dosimetry studies that focus on optimization of esophageal PDT.