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BACKGROUND: Re-excision rates following breast conserving surgery (BCS) remain as high as ~ 35%, with positive margins detected during follow-up histopathology. Additional breast cancer resection surgery is not only taxing on the patient and health care system, but also delays adjuvant therapies, increasing morbidity and reducing the likelihood of a positive outcome. The ability to precisely resect and visualize tumor margins in real time within the surgical theater would greatly benefit patients, surgeons and the health care system. Current tumor margin assessment technologies utilized during BCS involve relatively lengthy and labor-intensive protocols, which impede the surgical work flow. METHODS: In previous work, we have developed and validated a fluorescence imaging method termed dual probe difference specimen imaging (DDSI) to accurately detect benign and malignant tissue with direct correlation to the targeted biomarker expression levels intraoperatively. The DDSI method is currently on par with touch prep cytology in execution time (~ 15-min). In this study, the main goal was to shorten the DDSI protocol by decreasing tissue blocking and washing times to optimize the DDSI protocol to < 10-min whilst maintaining robust benign and malignant tissue differentiation. RESULTS: We evaluated the utility of the shortened DDSI staining methodology using xenografts grown from cell lines with varied epidermal growth factor receptor (EGFR) expression levels, comparing accuracy through receiver operator characteristic (ROC) curve analyses across varied tissue blocking and washing times. An optimized 8-min DDSI methodology was developed for future clinical translation. CONCLUSIONS: Successful completion of this work resulted in substantial shortening of the DDSI methodology for use in the operating room, that provided robust, highly receptor specific, sensitive diagnostic capabilities between benign and malignant tissues.
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Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Diagnóstico por Imagen/métodos , Sondas Moleculares , Animales , Neoplasias de la Mama/cirugía , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Estadificación de Neoplasias , Coloración y Etiquetado/métodosRESUMEN
Cherenkov emission induced by external beam radiation from a clinical linear accelerator has been shown in preclinical molecular imaging and clinical imaging. The broad spectrum Cherenkov emission should have a short wavelength infrared (SWIR, 1000-1700 nm) component, as predicted theoretically. To the best of our knowledge, this Letter is the first experimental observation of this SWIR Cherenkov emission induced by external beam radiation. The measured spectrum of SWIR Cherenkov emission matches the theoretical prediction, with a fluence rate near one-third of the visible and near-infrared red emissions (Vis-NIR, 400-900 nm). Imaging in water-based phantoms and biological tissues indicates that there is a sufficient fluence rate for radiotherapy dosimetry applications. The spatial resolution is improved approximately 5.3 times with SWIR Cherenkov emission detection versus Vis-NIR Cherenkov emission, which provides some improvement in the potential for higher resolution Cherenkov emission dosimetry and molecular sensing during clinical radiotherapy by imaging with SWIR wavelengths.
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BACKGROUND: Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) provides selective uptake and conversion of ALA into protoporphyrin IX (PpIX) in actinic keratosis and squamous cell carcinoma, yet large response variations in effect are common between individuals. The aim of this study was to compare pre-treatment strategies that increase the therapeutic effect, including fractionated light delivery during PDT (fPDT) and use of iron chelator desferrioxamine (DFO), separately and combined. METHODS: Optical measurements of fluorescence were used to quantify PpIX produced, and the total amount of PpIX photobleached as an implicit measure of the photodynamic dose. In addition, measurements of white light reflectance were used to quantify changes in vascular physiology throughout the PDT treatment. RESULTS: fPDT produced both a replenishment of PpIX and vascular re-oxygenation during a 2 h dark interval between the first and second PDT light fractions. The absolute photodynamic dose was increased 57% by fPDT, DFO and their combination, as compared with PDT group (from 0.7 to 1.1). Despite that light fractionation increased oedema and scab formation during the week after treatment, no significant difference in long-term survival has been observed between treatment groups. However, outcomes stratified on the basis of measured photodynamic dose showed a significant difference in long-term survival. CONCLUSIONS: The assessment of implicit photodynamic dose was a more significant predictor of efficacy for ALA-PDT skin cancer treatments than prescription of an enhanced treatment strategy, likely because of high individual variation in response between subjects.
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Ácido Aminolevulínico/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Deferoxamina/uso terapéutico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéutico , Protoporfirinas/uso terapéutico , Sideróforos/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Deferoxamina/farmacología , Fraccionamiento de la Dosis de Radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Hemo/biosíntesis , Humanos , Láseres de Semiconductores , Iluminación/instrumentación , Iluminación/métodos , Ratones , Ratones Desnudos , Fármacos Fotosensibilizantes/farmacocinética , Protoporfirinas/farmacocinética , Distribución Aleatoria , Sideróforos/farmacología , Neoplasias Cutáneas/patología , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.
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Fluorescencia , Glioma/metabolismo , Imagen por Resonancia Magnética/métodos , Imagen Molecular/métodos , Receptores de Superficie Celular/metabolismo , Tomografía Óptica/métodos , Animales , Receptores ErbB/metabolismo , Cinética , Ratones , Unión ProteicaRESUMEN
Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal. Aim: The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs. Approach: A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo. Results: In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice. Conclusions: We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.
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Citometría de Flujo , Células Neoplásicas Circulantes , Fantasmas de Imagen , Animales , Ratones , Células Neoplásicas Circulantes/patología , Citometría de Flujo/métodos , Línea Celular Tumoral , Humanos , Mieloma Múltiple/diagnóstico por imagen , Mieloma Múltiple/patología , Diseño de Equipo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genéticaRESUMEN
Assessing tumor margin status during surgery is critical to ensure complete resection of cancer tissue; however, current approaches are ineffective and often result in repeat surgery. We present an optical imaging approach for margin assessment using topical application of two fluorescent stains, one targeted to a tumor biomarker and the other a nontargeted reference, to freshly excised specimens. Computing a normalized difference image from fluorescence images of the targeted and untargeted stains suppresses the confounding effects of nonspecific uptake. Applying this approach in excised breast tumor models produced promising tumor-to-normal tissue contrasts that were significantly higher than single-targeted-stain imaging.
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Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/metabolismo , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/cirugía , Imagen Óptica/métodos , Tejido Adiposo/patología , Administración Tópica , Animales , Periodo Intraoperatorio , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Factores de TiempoRESUMEN
Radiation treatment of cancer induces an optical Cerenkov emission throughout the treated volume, which could be used to excite molecular reporters in vivo, allowing molecular sensing of tissue response during fractionated therapy. In this Letter, the idea that spatial mapping of this signal can be achieved with tomographic recovery of the fluorophore distribution is tested for the first time using 6 MV photons from a linear accelerator in a heterogeneous tissue phantom. Cerenkov light excited fluorophores throughout the tissue phantom, and diffuse tomography was used to recover images. Measurements from 13 locations were used, with spectrometer detection and spectral fitting, to separate the fluorophore emission from the Cerenkov continuum. Fluorescent diffuse tomographic images showed a linear response between the concentration and the reconstructed values. The potential to apply this molecular imaging in treatment with molecular reporters appears promising.
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Fluorescencia , Radioterapia/métodos , Tomografía/métodos , Fenómenos Ópticos , Fantasmas de Imagen , Radioterapia/instrumentación , Tomografía/instrumentaciónRESUMEN
Fluorescence guided surgery (FGS) is an emerging technology that has demonstrated improved surgical outcomes. However, dim lighting conditions required by current FGS systems are disruptive to standard surgical workflow. We present a novel FGS system capable of imaging fluorescence under normal room light by using pulsed excitation and gated acquisition. Images from tissue-simulating phantoms confirm visual detection down to 0.25 µM of protoporphyrin IX under 125 µW/cm2 of ambient light, more than an order of magnitude lower than that measured with the Zeiss Pentero in the dark. Resection of orthotopic brain tumors in mice also suggests that the pulsed-light system provides superior sensitivity in vivo.
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Fluorescencia , Luz , Iluminación , Cirugía Asistida por Computador/métodos , Animales , Glioma/cirugía , RatonesRESUMEN
Since its discovery during the 1930s the Cerenkov effect (light emission from charged particles traveling faster than the local speed of light in a dielectric medium) has been paramount in the development of high-energy physics research. The ability of the emitted light to describe a charged particle's trajectory, energy, velocity, and mass has allowed scientists to study subatomic particles, detect neutrinos, and explore the properties of interstellar matter. However, to our knowledge, all applications of the process to date have focused on the identification of particles themselves, rather than their effect upon the surroundings through which they travel. Here we explore a novel application of the Cerenkov effect for the recovery of the spatial distribution of ionizing radiation energy deposition in a medium and apply it to the issue of dose determination in medical physics. By capturing multiple projection images of the Cerenkov light induced by a medical linear accelerator x-ray photon beam, we demonstrate the successful three-dimensional tomographic reconstruction of the imparted dose distribution.
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Imagenología Tridimensional/métodos , Tomografía/métodos , RadiometríaRESUMEN
Neurosurgical fluorescence guidance relies on contrast agents to identify tumor regions to aid in increasing the extent of resection. Existing contrast agents for this indication each have their own limitation: unpredictable contrast from tumor heterogeneity, significant extravasation into the background brain and long incubation times. An ideal contrast agent should have high and rapid contrast that persists well into the surgical procedure. By using a whole animal hyperspectral cryo-imaging system several CAs were screened for these favorable properties and compared to the gold standard of gadolinium enhanced MR. Herein, we briefly report on the leading candidate Rd-PEG1k, which shows high contrast within minutes of administration that persists for at least 90 minutes.
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Significance: Positive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative. Aim: Current intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients. Approach: Dual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images (i.e., DDSI) of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer. Results: Targeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues (prostate, adipose, muscle). Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues. Conclusions: Previous work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker.
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Neoplasias de la Próstata , Humanos , Masculino , Diagnóstico por Imagen , Próstata/diagnóstico por imagen , Próstata/cirugía , Próstata/patología , Prostatectomía , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/cirugíaRESUMEN
PURPOSE: Reliable and rapid identification of tumor in the margins of breast specimens during breast-conserving surgery to reduce repeat surgery rates is an active area of investigation. Dual-stain difference imaging (DDSI) is one of many approaches under evaluation for this application. This technique aims to topically apply fluorescent stain pairs (one targeted to a receptor-of-interest and the other a spectrally distinct isotype), image both stains, and compute a normalized difference image between the two channels. Prior evaluation and optimization in a variety of preclinical models produced encouraging diagnostic performance. Herein, we report on a pilot clinical study which evaluated HER2-targeted DDSI on 11 human breast specimens. PROCEDURES: Gross sections from 11 freshly excised mastectomy specimens were processed using a HER2-receptor-targeted DDSI protocol shortly after resection. After staining with the dual-probe protocol, specimens were imaged on a fluorescence scanner, followed by tissue fixation for hematoxylin and eosin and anti-HER2 immunohistochemical staining. Receiver operator characteristic curves and area under the curve (AUC) analysis were used to assess diagnostic performance of the resulting images. Performance values were also compared to expression level determined from IHC staining. RESULTS: Eight of the 11 specimens presented with distinguishable invasive ductal carcinoma and/or were not affected by an imaging artifact. In these specimens, the DDSI technique provided an AUC = 0.90 ± 0.07 for tumor-to-adipose tissue and 0.81 ± 0.15 for tumor-to-glandular tissue, which was significantly higher than AUC values recovered from images of the targeted probe alone. DDSI values and diagnostic performance did not correlate with HER2 expression level, and tumors with low HER2 expression often produced high AUC, suggesting that even the low expression levels were enough to help distinguish tumor. CONCLUSIONS: The results from this preliminary study of rapid receptor-specific staining in human specimens were consistent with prior preclinical results and demonstrated promising diagnostic potential.
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Neoplasias de la Mama , Mastectomía , Humanos , Femenino , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/cirugía , Mastectomía Segmentaria , Colorantes , Coloración y EtiquetadoRESUMEN
Hyperspectral imaging and spectral analysis quantifies fluorophore concentration during fluorescence-guided surgery1-6. However, acquisition of the multiple wavelengths required to implement these methods can be time-consuming and hinder surgical workflow. To this end, a snapshot hyperspectral imaging system capable of acquiring 64 channels of spectral data simultaneously was developed for rapid hyperspectral imaging during neurosurgery. The system uses a birefringent spectral demultiplexer to split incoming light and redirect wavelengths to different sections of a large format microscope sensor. Its configuration achieves high optical throughput, accepts unpolarized input light and exceeds channel count of prior image-replicating imaging spectrometers by 4-fold. Tissue-simulating phantoms consisting of serial dilutions of the fluorescent agent characterize system linearity and sensitivity, and comparisons to performance of a liquid crystal tunable filter based hyperspectral imaging device are favorable. The new instrument showed comparable, if not improved, sensitivity at low fluorophore concentrations; yet, acquired wide-field images at more than 70-fold increase in frame rate. Image data acquired in the operating room during human brain tumor resection confirm these findings. The new device is an important advance in achieving real-time quantitative imaging of fluorophore concentration for guiding surgery.
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Neoplasias Encefálicas , Neurocirugia , Humanos , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/cirugía , Protoporfirinas , Imagen Óptica/métodos , Colorantes FluorescentesRESUMEN
Radiation from a linear accelerator induces Cherenkov emission in tissue, which has recently been shown to produce biochemical spectral signatures that can be interpreted to estimate tissue hemoglobin and oxygen saturation or molecular fluorescence from reporters. The Cherenkov optical light levels are in the range of 10(-6) to 10(-9) W/cm2, which limits the practical utility of the signal in routine radiation therapy monitoring. However, due to the fact that the radiation is pulsed, gated-acquisition of the signal allows detection in the presence of ambient lighting, as is demonstrated here. This observation has the potential to significantly increase the value of Cherenkov emission spectroscopy during radiation therapy to monitor tissue molecular events.
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Dosimetría por Película/instrumentación , Monitoreo Fisiológico/instrumentación , Neoplasias/radioterapia , Oxígeno/análisis , Análisis Espectral/instrumentación , Diseño de Equipo , Hemoglobinas/análisis , Humanos , Aceleradores de Partículas , Fantasmas de Imagen , Dispersión de RadiaciónRESUMEN
PURPOSE: The ability to noninvasively quantify receptor availability (RA) in solid tumors is an aspirational goal of molecular imaging, often challenged by the influence of non-specific accumulation of the contrast agent. Paired-agent imaging (PAI) techniques aim to compensate for this effect by imaging the kinetics of a targeted agent and an untargeted isotype, often simultaneously, and comparing the kinetics of the two agents to estimate RA. This is usually accomplished using two spectrally distinct fluorescent agents, limiting the technique to superficial tissues and/or preclinical applications. Applying the approach in humans using conventional imaging modalities is generally infeasible since most modalities are unable to routinely image multiple agents simultaneously. We examine the ability of PAI to be implemented in a cross-modality paradigm, in which the targeted and untargeted agent kinetics are imaged with different modalities and used to recover receptor availability. PROCEDURES: Eighteen mice bearing orthotopic brain tumors were administered a solution containing three contrast agents: (1) a fluorescent agent targeted to epidermal growth factor receptor (EGFR), (2) an untargeted fluorescent isotype, and (3) a gadolinium-based contrast agent (GBCA) for MRI imaging. The kinetics of all three agents were imaged for 1 h after administration using an MRI-coupled fluorescence tomography system. Paired-agent receptor availability was computed using (1) the conventional all-optical approach using the targeted and untargeted optical agent images and (2) the cross-modality approach using the targeted optical and untargeted MRI-GBCA images. Receptor availability estimates between the two methods were compared. RESULTS: Receptor availability values using the cross-modality approach were highly correlated to the conventional, single-modality approach (r = 0.94; p < 0.00001). CONCLUSION: These results suggest that cross-modality paired-agent imaging for quantifying receptor availability is feasible. Ultimately, cross-modality paired-agent imaging could facilitate rapid, noninvasive receptor availability quantification in humans using hybrid clinical imaging modalities.
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Neoplasias Encefálicas , Animales , Medios de Contraste , Estudios de Factibilidad , Imagen por Resonancia Magnética , Ratones , Imagen Molecular/métodosRESUMEN
Neonatal herpes simplex virus (nHSV) infections often result in significant mortality and neurological morbidity despite antiviral drug therapy. Maternally transferred herpes simplex virus (HSV)-specific antibodies reduce the risk of clinically overt nHSV, but this observation has not been translationally applied. Using a neonatal mouse model, we tested the hypothesis that passive transfer of HSV-specific human mAbs can prevent mortality and morbidity associated with nHSV. The mAbs were expressed in vivo via vectored immunoprophylaxis or recombinantly. Through these maternally derived routes or through direct administration to pups, diverse mAbs to HSV glycoprotein D protected against neonatal HSV-1 and HSV-2 infection. Using in vivo bioluminescent imaging, both pre- and post-exposure mAb treatment significantly reduced viral load in mouse pups. Together these studies support the notion that HSV-specific mAb-based therapies could prevent or improve HSV infection outcomes in neonates.
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Herpes Simple , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales , Antivirales , Glicoproteínas , Humanos , Ratones , Morbilidad , Complicaciones Infecciosas del EmbarazoRESUMEN
PURPOSE: Cerenkov emission is induced when a charged particle moves faster than the speed of light in a given medium. Both x-ray photons and electrons produce optical Cerenkov photons in everyday radiation therapy of tissue; yet, this phenomenon has never been fully documented. This study quantifies the emissions and also demonstrates that the Cerenkov emission can excite a fluorophore, protoporphyrin IX (PpIX), embedded in biological phantoms. METHODS: In this study, Cerenkov emission induced by radiation from a clinical linear accelerator is investigated. Biological mimicking phantoms were irradiated with x-ray photons, with energies of 6 or 18 MV, or electrons at energies 6, 9, 12, 15, or 18 MeV. The Cerenkov emission and the induced molecular fluorescence were detected by a camera or a spectrometer equipped with a fiber optic cable. RESULTS: It is shown that both x-ray photons and electrons, at MeV energies, produce optical Cerenkov photons in tissue mimicking media. Furthermore, we demonstrate that the Cerenkov emission can excite a fluorophore, protoporphyrin IX (PpIX), embedded in biological phantoms. CONCLUSIONS: The results here indicate that molecular fluorescence monitoring during external beam radiotherapy is possible.
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Mediciones Luminiscentes/métodos , Técnicas de Sonda Molecular , Radiometría/métodos , Radioterapia Conformacional/métodos , Fluorescencia , Dosis de RadiaciónRESUMEN
Concurrent administration of cancer therapeutics with tumor vasculature targeting treatment has been shown to improve overall survival in multiple human cancer types, as such combinations aim to destroy different compartments of tumors. Anti-angiogenesis therapeutics designed to inhibit tumor induced vessel sprouting have also been shown to re-model the tumor vasculature through a transient vessel normalization effect, which leads to improved perfusion of oxygen and drug in tumor. However, the effects that this normalized vasculature has on the availability of cancer receptor, such as EGFR, is unknown. Herein, we examined the use of MRI-PAFT to estimate cancer surface receptor availability in response to anti-angiogenesis therapy, using MRI-coupled paired agent fluorescence tomography. Bevacizumab treated tumors showed increase in RA compared to control tumors, but this was not statistically significant.
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Fluorescent contrast agents targeted to cancer biomarkers are increasingly being explored for cancer detection, surgical guidance, and response monitoring. Efforts have been underway to topically apply such biomarker-targeted agents to freshly excised specimen for detecting cancer cell receptors on the surface as a method for intraoperative surgical margin assessment, including dual-probe staining methods introduce a second 'non-specific' optical agent as a control to help compensate for heterogeneous uptake and normalize the imaging field. Still, such specimen staining protocols introduce multifaceted complexity with unknown variables, such as tissue-specific diffusion, cell-specific binding and disassociation rates, and other factors, affecting the interpreted cancer-biomarker distribution across the specimen surface. The ability to recover three-dimensional dual-probe biodistributions throughout whole-specimens could offer a ground-truth validation method for examining topical staining uptake behaviors. Herein, we report on a novel method for characterizing dual-probe accumulation with 3D depth-profiles observed from a dual-probe fresh-specimen staining experiment.
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Whole-animal fluorescence cryo-imaging is an established technique that enables visualization of the biodistribution of labeled drugs, contrast agents, functional reporters and cells in detail. However, many tissues produce endogenous autofluorescence, which can confound interpretation of the cryo-imaging volumes. We describe a multi-channel, hyperspectral cryo-imaging system that acquires densely-sampled spectra at each pixel in the 3-dimensional stack. This information enables the use of spectral unmixing to isolate the fluorophore-of-interest from autofluorescence and/or other fluorescent reporters. In phantoms and a glioma xenograft model, we show that the approach improves detection limits, increases tumor contrast, and can dramatically alter image interpretation.