Combination of an Integrin-Targeting NIR Tracer and an Ultrasensitive Spectroscopic Device for Intraoperative Detection of Head and Neck Tumor Margins and Metastatic Lymph Nodes.

Despite major advances in targeted drug therapy and radiation therapy, surgery remains the most effective treatment for most solid tumors. The single most important predictor of patient survival is a complete surgical resection of the primary tumor, draining lymph nodes, and metastatic lesions. Presently, however, 20%-30% of patients with head and neck cancer who undergo surgery still leave the operating room without complete resection because of missed lesions. Thus, major opportunities exist to develop advanced imaging tracers and intraoperative instrumentation that would allow surgeons to visualize microscopic tumors during surgery. The cell adhesion molecule integrin αvß3 is specifically expressed by tumor neovasculature and invading tumor cells, but not by quiescent vessels or normal cells. Here we report the combined use of an integrin-targeting near-infrared tracer (RGD-IRDye800CW) and a handheld spectroscopic device, an integrated point spectroscopy with wide-field imaging system, for highly sensitive detection of integrin overexpression on infiltrating cancer cells. By using an orthotopic head and neck cancer animal model, we show that this tracer-device combination allows intraoperative detection of not only invasive tumor margins but also metastatic lymph nodes. Correlated histological analysis further reveals that microscopic clusters of 50-100 tumor cells can be detected intraoperatively with high sensitivity and specificity, raising new possibilities in guiding surgical resection of microscopic tumors and metastatic lymph nodes.

An integrated widefield imaging and spectroscopy system for contrast-enhanced, image-guided resection of tumors.

UNLABELLED: Tumor recurrence following surgery is a common and unresolved medical problem of great importance since surgery is the most widely used treatment for solid-mass tumors worldwide. A contributing factor to tumor recurrence is the presence of residual tumor remaining at or near the surgical site following surgery. GOAL: The primary objective of this study was to develop and evaluate an image-guided surgery system based on a near-infrared, handheld excitation source and spectrograph in combination with a widefield video imaging system. METHODS: This system was designed to detect the fluorescence of near-infrared contrast agents and, in particular, indocyanine green (ICG). The imaging system was evaluated for its optical performance and ability to detect the presence of ICG in tumors in an ectopic murine tumor model as well as in spontaneous tumors arising in canines. RESULTS: In both settings, an intravenous ICG infusion provided tumor contrast. In both the murine models and surgical specimens from canines, ICG preferentially accumulated in tumor tissue compared to surrounding normal tissue. The resulting contrast was sufficient to distinguish neoplasia from normal tissue; in the canine surgical specimens, the contrast was sufficient to permit identification of neoplasia on the marginal surface of the specimen. CONCLUSION: These results demonstrate a unique concept in image-guided surgery by combining local excitation and spectroscopy with widefield imaging. SIGNIFICANCE: The ability to readily detect ICG in canines with spontaneous tumors in a clinical setting exemplifies the potential for further clinical translation; the promising results of detecting neoplasia on the marginal specimen surface underscore the clinical utility.

Assessment of intra-observer variability in measurement of high-grade brain tumors.

A 25% increase in bidimensional products (BPs) of tumor diameter has been used as a criterion for brain tumor progression. We studied intra-observer variability in measurements of BPs. Ten patients with contrast-enhancing glioblastoma multiforme underwent baseline and follow-up MR imaging. Seven observers measured BPs in various planes. Differences in BPs between scans were expressed as a percentage of baseline. This calculation was performed for both readings of the baseline and follow-up scans. Differences between change from baseline to follow-up on each reading (termed D values) were calculated for each reader (total of 196 D values). Median D value in each plane was calculated for each reader. Range of D values was 12.36-33.64% in axial plane (average 10.63%), 12.18-38.62% in coronal plane (average 26.84%) and 15.12-35.48% in sagittal plane (average 26.11%). Across all planes, 88 (45%) D values were >25%. When all imaging planes for any single observation were combined, in 76% of cases, at least one D value of >25% was seen. Based on the high degree of intra-observer variability, tumor measurements producing an increase in BP of >25% can routinely be obtained solely by chance.

Hand-held spectroscopic device for in vivo and intraoperative tumor detection: contrast enhancement, detection sensitivity, and tissue penetration.

Surgery is one of the most effective and widely used procedures in treating human cancers, but a major problem is that the surgeon often fails to remove the entire tumor, leaving behind tumor-positive margins, metastatic lymph nodes, and/or satellite tumor nodules. Here we report the use of a hand-held spectroscopic pen device (termed SpectroPen) and near-infrared contrast agents for intraoperative detection of malignant tumors, based on wavelength-resolved measurements of fluorescence and surface-enhanced Raman scattering (SERS) signals. The SpectroPen utilizes a near-infrared diode laser (emitting at 785 nm) coupled to a compact head unit for light excitation and collection. This pen-shaped device effectively removes silica Raman peaks from the fiber optics and attenuates the reflected excitation light, allowing sensitive analysis of both fluorescence and Raman signals. Its overall performance has been evaluated by using a fluorescent contrast agent (indocyanine green, or ICG) as well as a surface-enhanced Raman scattering (SERS) contrast agent (pegylated colloidal gold). Under in vitro conditions, the detection limits are approximately 2-5 × 10(-11) M for the indocyanine dye and 0.5-1 × 10(-13) M for the SERS contrast agent. Ex vivo tissue penetration data show attenuated but resolvable fluorescence and Raman signals when the contrast agents are buried 5-10 mm deep in fresh animal tissues. In vivo studies using mice bearing bioluminescent 4T1 breast tumors further demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence. After surgery, the SpectroPen device permits further evaluation of both positive and negative tumor margins around the surgical cavity, raising new possibilities for real-time tumor detection and image-guided surgery.

Oxidative quenching and degradation of polymer-encapsulated quantum dots: new insights into the long-term fate and toxicity of nanocrystals in vivo.

We report quenching and chemical degradation of polymer-coated quantum dots by reactive oxygen species (ROS), a group of oxygen-containing molecules that are produced by cellular metabolism and are involved in both normal physiological and disease processes such as oxidative signaling, cancer, and atherosclerosis. A major new finding is that hypochlorous acid (HOCl) in its neutral form is especially potent in degrading encapsulated QDs, due to its small size, neutral charge, long half-life, and fast reaction kinetics under physiologic conditions. Thus, small and neutral molecules such as HOCl and hydrogen peroxide (H2O2) are believed to diffuse across the polymer coating layer, leading to chemical oxidation of sulfur or selenium atoms on the QD surface. This "etching" process first generates lattice structural defects (which cause fluorescence quenching) and then produces soluble metal (e.g., cadmium and zinc) and chalcogenide (e.g., sulfur and selenium) species. We also find that significant fluorescence quenching occurs before QD dissolution and that localized surface defects can be repaired or "annealed" by UV light illumination. These results have important implications regarding the long-term fate and potential toxicity of semiconductor nanocrystals in vivo.

Minimizing nonspecific cellular binding of quantum dots with hydroxyl-derivatized surface coatings.

Quantum-dot (QD) nanocrystals are promising fluorescent probes for multiplexed staining assays in biological applications. However, nonspecific QD binding to cellular membranes and proteins remains a limiting factor in detection sensitivity and specificity. Here we report a new class of hydroxyl (-OH)-coated QDs for minimizing nonspecific cellular binding and for overcoming the bulky size problems encountered with previous surface coatings. The hydroxylated QDs are prepared from carboxylated (-COOH) dots via a hydroxylation and cross-linking process. With a compact hydrodynamic size of 13-14 nm (diameter), they are highly fluorescent (>60% quantum yields) and stable under both basic and acidic conditions. By using human cancer cells, we have evaluated their superior nonspecific binding properties against that of carboxylated, protein-coated, and poly(ethylene glycol) (PEG)-coated QDs. Quantitative cellular staining data indicate that the hydroxylated QDs result in a dramatic 140-fold reduction in nonspecific binding relative to that of carboxylated dots and a still significant 10-20-fold reduction relative to that of PEG- and protein-coated dots.