Kenneth M. Tichauer

Quantitative in vivo cell-surface receptor imaging in oncology: kinetic modeling and paired-agent principles from nuclear medicine and optical imaging

The development of methods to accurately quantify cell-surface receptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surface receptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surface receptor concentrations present in tissue, although each likely has a different clinical niche.

Quantitative In Vivo Immunohistochemistry of Epidermal Growth Factor Receptor Using a Receptor Concentration Imaging Approach

As receptor-targeted therapeutics become increasingly used in clinical oncology, the ability to quantify protein expression and pharmacokinetics in vivo is imperative to ensure successful individualized treatment plans. Current standards for receptor analysis are performed on extracted tissues. These measurements are static and often physiologically irrelevant; therefore, only a partial picture of available receptors for drug targeting in vivo is provided. Until recently, in vivo measurements were limited by the inability to separate delivery, binding, and retention effects, but this can be circumvented by a dual-tracer approach for referencing the detected signal. We hypothesized that in vivo receptor concentration imaging (RCI) would be superior to ex vivo immunohistochemistry (IHC). Using multiple xenograft tumor models with varying EGFR expression, we determined the EGFR concentration in each model using a novel targeted agent (anti-EGFR affibody-IRDye800CW conjugate) along with a simultaneously delivered reference agent (control affibody-IRDye680RD conjugate). The RCI-calculated in vivo receptor concentration was strongly correlated with ex vivo pathologist-scored IHC and computer-quantified ex vivo immunofluorescence. In contrast, no correlation was observed with ex vivo Western blot analysis or in vitro flow-cytometry assays. Overall, our results argue that in vivo RCI provides a robust measure of receptor expression equivalent to ex vivo immunostaining, with implications for use in noninvasive monitoring of therapy or therapeutic guidance during surgery.

Quantification of the binding potential of cell-surface receptors in fresh excised specimens via dual-probe modeling of SERS nanoparticles

The complete removal of cancerous tissue is a central aim of surgical oncology, but is difficult to achieve in certain cases, especially when the removal of surrounding normal tissues must be minimized. Therefore, when post-operative pathology identifies residual tumor at the surgical margins, re-excision surgeries are often necessary. An intraoperative approach for tumor-margin assessment, insensitive to nonspecific sources of molecular probe accumulation and contrast, is presented employing kinetic-modeling analysis of dual-probe staining using surface-enhanced Raman scattering nanoparticles (SERS NPs). Human glioma (U251) and epidermoid (A431) tumors were implanted subcutaneously in six athymic mice. Fresh resected tissues were stained with an equimolar mixture of epidermal growth factor receptor (EGFR)-targeted and untargeted SERS NPs. The binding potential (BP; proportional to receptor concentration) of EGFR – a cell-surface receptor associated with cancer – was estimated from kinetic modeling of targeted and untargeted NP concentrations in response to serial rinsing. EGFR BPs in healthy, U251, and A431 tissues were 0.06 ± 0.14, 1.13 ± 0.40, and 2.23 ± 0.86, respectively, which agree with flow-cytometry measurements and published reports. The ability of this approach to quantify the BP of cell-surface biomarkers in fresh tissues opens up an accurate new approach to analyze tumor margins intraoperatively.

Biomaterials with persistent growth factor gradients in vivo accelerate vascularized tissue formation

Gradients of soluble factors play an important role in many biological processes, including blood vessel assembly. Gradients can be studied in detail inxa0vitro, but methods that enable the study of spatially distributed soluble factors and multi-cellular processes inxa0vivo are limited. Here, we report on a method for the generation of persistent inxa0vivo gradients of growth factors in a three-dimensional (3D) biomaterial system. Fibrin loaded porous poly (ethylene glycol) (PEG) scaffolds were generated using a particulate leaching method. Platelet derived growth factor BB (PDGF-BB) was encapsulated into poly (lactic-co-glycolic acid) (PLGA) microspheres which were placed distal to the tissue-material interface. PLGA provides sustained release of PDGF-BB and its diffusion through the porous structure results in gradient formation. Gradients within the scaffold were confirmed inxa0vivo using near-infrared fluorescence imaging and gradients were present for more than 3 weeks. The diffusion of PDGF-BB was modeled and verified with inxa0vivo imaging findings. The depth of tissue invasion and density of blood vessels formed in response to the biomaterial increased with magnitude of the gradient. This biomaterial system allows for generation of sustained growth factor gradients for the study of tissue response to gradients inxa0vivo.

Accounting for pharmacokinetic differences in dual-tracer receptor density imaging.

Dual-tracer molecular imaging is a powerful approach to quantify receptor expression in a wide range of tissues by using an untargeted tracer to account for any nonspecific uptake of a molecular-targeted tracer. This approach has previously required the pharmacokinetics of the receptor-targeted and untargeted tracers to be identical, requiring careful selection of an ideal untargeted tracer for any given targeted tracer. In this study, methodology capable of correcting for tracer differences in arterial input functions, as well as binding-independent delivery and retention, is derived and evaluated in a mouse U251 glioma xenograft model using an Affibody tracer targeted to epidermal growth factor receptor (EGFR), a cell membrane receptor overexpressed in many cancers. Simulations demonstrated that blood, and to a lesser extent vascular-permeability, pharmacokinetic differences between targeted and untargeted tracers could be quantified by deconvolving the uptakes of the two tracers in a region of interest devoid of targeted tracer binding, and therefore corrected for, by convolving the uptake of the untargeted tracer in all regions of interest by the product of the deconvolution. Using fluorescently labeled, EGFR-targeted and untargeted Affibodies (known to have different blood clearance rates), the average tumor concentration of EGFR in four mice was estimated using dual-tracer kinetic modeling to be 3.9 ± 2.4xa0nM compared to an expected concentration of 2.0 ± 0.4xa0nM. However, with deconvolution correction a more equivalent EGFR concentration of 2.0 ± 0.4xa0nM was measured.

Tumor Endothelial Marker Imaging in Melanomas Using Dual-Tracer Fluorescence Molecular Imaging

PurposeCancer-specific endothelial markers available for intravascular binding are promising targets for new molecular therapies. In this study, a molecular imaging approach of quantifying endothelial marker concentrations (EMCI) is developed and tested in highly light-absorbing melanomas. The approach involves injection of targeted imaging tracer in conjunction with an untargeted tracer, which is used to account for nonspecific uptake and tissue optical property effects on measured targeted tracer concentrations.ProceduresTheoretical simulations and a mouse melanoma model experiment were used to test out the EMCI approach. The tracers used in the melanoma experiments were fluorescently labeled anti-Plvap/PV1 antibody (plasmalemma vesicle associated protein Plvap/PV1 is a transmembrane protein marker exposed on the luminal surface of endothelial cells in tumor vasculature) and a fluorescent isotype control antibody, the uptakes of which were measured on a planar fluorescence imaging system.ResultsThe EMCI model was found to be robust to experimental noise under reversible and irreversible binding conditions and was capable of predicting expected overexpression of PV1 in melanomas compared to healthy skin despite a 5-time higher measured fluorescence in healthy skin compared to melanoma: attributable to substantial light attenuation from melanin in the tumors.ConclusionsThis study demonstrates the potential of EMCI to quantify endothelial marker concentrations in vivo, an accomplishment that is currently unavailable through any other methods, either in vivo or ex vivo.

Perfusion CT estimates photosensitizer uptake and biodistribution in a rabbit orthotopic pancreatic cancer model: a pilot study.

RATIONALE AND OBJECTIVESnIt was hypothesized that perfusion computed tomography (CT), blood flow (BF), blood volume (BV), and vascular permeability surface area (PS) product parameters would be predictive of therapeutic anticancer agent uptake in pancreatic cancer, facilitating image-guided interpretation of human treatments. The hypothesis was tested in an orthotopic rabbit model of pancreatic cancer, by establishing the model, imaging with endoscopic ultrasound (EUS) and contrast CT, and spatially comparing the perfusion maps to the ex vivo uptake values of the injected photosensitizer, verteporfin.nnnMATERIALS AND METHODSnNine New Zealand white rabbits underwent direct pancreas implantation of VX2 tumors, and CT perfusion or EUS was performed 10 days postimplantation. Verteporfin was injected during CT imaging, and the tissue was removed 1 hour postinjection for frozen tissue fluorescence scanning. Region-of-interest comparisons of CT data with ex vivo fluorescence and histopathologic staining were performed.nnnRESULTSnDynamic contrast-enhanced CT showed enhanced BF, BV, and PS in the tumor rim and decreased BF, BV, and PS in the tumor core. Significant correlations were found between ex vivo verteporfin concentration and each of BF, BV, and PS.nnnCONCLUSIONSnThe efficacy of verteporfin delivery in tumors is estimated by perfusion CT, providing a noninvasive method of mapping photosensitizer dose.

Pixel-based absorption correction for dual-tracer fluorescence imaging of receptor binding potential.

Ratiometric approaches to quantifying molecular concentrations have been used for decades in microscopy, but have rarely been exploited in vivo until recently. One dual-tracer approach can utilize an untargeted reference tracer to account for non-specific uptake of a receptor-targeted tracer, and ultimately estimate receptor binding potential quantitatively. However, interpretation of the relative dynamic distribution kinetics is confounded by differences in local tissue absorption at the wavelengths used for each tracer. This study simulated the influence of absorption on fluorescence emission intensity and depth sensitivity at typical near-infrared fluorophore wavelength bands near 700 and 800 nm in mouse skin in order to correct for these tissue optical differences in signal detection. Changes in blood volume [1-3%] and hemoglobin oxygen saturation [0-100%] were demonstrated to introduce substantial distortions to receptor binding estimates (error > 30%), whereas sampled depth was relatively insensitive to wavelength (error < 6%). In response, a pixel-by-pixel normalization of tracer inputs immediately post-injection was found to account for spatial heterogeneities in local absorption properties. Application of the pixel-based normalization method to an in vivo imaging study demonstrated significant improvement, as compared with a reference tissue normalization approach.

Direct characterization of arterial input functions by fluorescence imaging of exposed carotid artery to facilitate kinetic analysis.

PurposeWith the goal of facilitating tracer kinetic analysis in small-animal planar fluorescence imaging, an experimental method for characterizing tracer arterial input functions is presented. The proposed method involves exposing the common carotid arteries by surgical dissection, which can then be imaged directly during tracer injection and clearance.ProceduresArterial concentration curves of IRDye-700DX-carboxylate, IRDye-800CW-EGF, and IRDye-800CW conjugated to anti-EGFR Affibody are recovered from athymic female mice (nu2009=u200912) by directly imaging exposed vessels. Images were acquired with two imaging protocols: a slow-kinetics approach (temporal resolutionu2009=u200945xa0s) to recover the arterial curves from two tracers simultaneously, and a fast-kinetics approach (temporal resolutionu2009=u2009500xa0ms) to characterize the first-pass peak of a single tracer. Arterial input functions obtained by the carotid imaging technique, as well as plasma curves measured by blood sampling were fit with a biexponential pharmacokinetic model.ResultsPharmacological fast- and slow-phase rate constants recovered with the proposed method were 0.37u2009±u20090.26 and 0.007u2009±u20090.001xa0min−1, respectively, for the IRDye700DX-C. For the IRDye800CW-EGF, the rate constants were 0.11u2009±u20090.13 and 0.003u2009±u20090.002xa0min−1. These rate constants did not differ significantly from those calculated previously by blood sampling, as determined by an F test; however, the between-subject variability was four times lower for arterial curves recovered using the proposed technique, compared with blood sampling.ConclusionsThe proposed technique enables the direct characterization of arterial input functions for kinetic analysis. As this method requires no additional instrumentation, it is immediately deployable in commercially available planar fluorescence imaging systems.

Nodal lymph flow quantified with afferent vessel input function allows differentiation between normal and cancer-bearing nodes.

Morbidity and complexity involved in lymph node staging via surgical resection and biopsy could ideally be improved using node assay techniques that are non-invasive. While visible blue dyes are often used to locate the sentinel lymph nodes from draining lymphatic vessels near a tumor, they do not provide an in situ metric to evaluate presence of cancer. In this study, the transport kinetics of methylene blue were analyzed to determine the potential for better in situ information about metastatic involvement in the nodes. A rat model with cancer cells in the axillary lymph nodes was used, with methylene blue injection to image the fluorescence kinetics. The lymphatic flow from injection sites to nodes was imaged and the relative kinetics from feeding lymphatic ducts relative to lymph nodes was quantified. Large variability existed in raw fluorescence and transport patterns within each cohort resulting in no systematic difference between average nodal uptake in normal, sham control and cancer-bearing nodes. However, when the signal from the afferent lymph vessel fluorescence was used to normalize the signal of the lymph nodes, the high signal heterogeneity was reduced. Using a model, the lymph flow through the nodes [Formula: see text] was estimated to be 1.49 ± 0.64 ml/g/min in normal nodes, 1.53 ± 0.45 ml/g/min in sham control nodes, and reduced to 0.50 ± 0.24 ml/g/min in cancer-cell injected nodes. This summarizes the significant difference (p = 0.0002) between cancer-free and cancer-bearing nodes in normalized flow. This process of normalized flow imaging could be used as an in situ tool to detect metastatic involvement in nodes.