Michael C. Mancini

Bioimaging: Second window for in vivo imaging

Enhanced fluorescence from carbon nanotubes and advances in near-infrared cameras have opened up a new wavelength window for small animal imaging.

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.

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.

Second window for in vivo imaging

Enhanced fluorescence from carbon nanotubes and advances in near-infrared cameras have opened up a new wavelength window for small animal imaging.

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.

An Integrated Widefield Imaging and Spectroscopy System for Contrast-Enhanced, Image-Guided Resection of Tumors

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.

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 αvβ3 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.

Abstract 2257: Optical imaging guidance of tumor resection margin for head and neck cancer

Traditional methods for assessing adequacy of head and neck tumor resection rely upon intraoperative palpation or visualization. Positive margins are strongly associated with recurrence and poor patient survival. To ensure the complete tumor removal, surgeons may resect additional peritumoral tissues. During the past decade, intraoperative pathologic consultation by frozen section has become standard. Each round of consultation takes 20 minutes and the average number of such consultation is 3 − 4 per operation, which prolongs the procedure. Two additional potential problems are: (1) it is impossible to locate all cohorts of infiltrating tumor cells; (2) the final pathology may reveal a positive margin when the frozen section was negative. Currently 20 − 30% of head and neck patients that undergo surgery still leave the operating room with an incomplete resection. Thus, major opportunities exist to develop a deep tissue imaging near infrared (NIR) agent and an intraoperative instrumentation that would allow surgeons to visualize microscopic tumors during surgery. The advantage of using optical imaging over radioisotopes is that it avoids continuous exposure of operating room personnel to radioactivity. The cell adhesion molecule integrin αvβ3 is specifically expressed by tumor neovasculature and invading tumor cells, but not by quiescent vessels or normal cells. We exploited integrin overexpression on infiltrating cancer cells for intraoperative detection of tumor margins. We tested a new optical instrumentation in conjunction with the integrin-targeted NIR dye, RGD-IRDye800CW, for assessing malignant tumor margins in an orthotopic head and neck cancer animal model. RGD peptides have high binding specificity to integrin αvβ3. For intraoperative tumor margin detection, we developed an intraoperative wavelength-resolved NIR imaging system. This device offers real-time, portable detection of tumor margins with high stability, faster throughput, and better co-registration than other imaging methods. In our animal model, the tumors infiltrated to the mylohoid neck muscle beyond tumor boundaries and the primary tumor resection left residual infiltrating tumors and metastatic lymph nodes behind, which were easily detected by our technology. We also found NIR signal in several spots of the lungs from the same mice. Histological analysis in correlation to imaging results support that we can detect extremely small cohorts of 20 − 50 tumor cells in intraoperative settings with high specificity. Thus our technology will be ideal in detecting tumor margins intraoperatively because the average number of tumor cells in invading cohorts is 50 − 100 and NIR penetration depth is about 3 − 5 cm. Of note, we found that NIR signals from the RGD peptides were stronger at the edge (rim) of tumors than the center; therefore, the integrin-targeted imaging probe will allow us to detect the tumor margins as well as infiltrating cohorts of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2257.

Second window for in vivo imaging: Bioimaging

Enhanced fluorescence from carbon nanotubes and advances in near-infrared cameras have opened up a new wavelength window for small animal imaging.