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Objectives Tumour-positive resection margins are a major problem during oral cancer surgery. gGlu-HMRG is a tracer that becomes fluorescent upon activation by gamma-glutamyltranspeptidase (GGT). This study aims to investigate the combination of gGlu-HMRG and a clinical fluorescence imaging system for the detection of tumour-positive resection margins. Materials and methods The preclinical Maestro and clinical Artemis imaging systems were compared in vitro and ex vivo with cultured human head and neck cancer cells (OSC19, GGT-positive; and FaDu, GGT negative) and tumour-bearing nude mice. Subsequently, frozen sections of normal and oral cancer tissues were ex vivo sprayed with gGlu-HMRG to determine the sensitivity and specificity. Finally, resection margins of patients with suspected oral cancer were ex vivo sprayed with gGlu-HMRG to detect tumour-positive resection margins. Results Both systems could be used to detect gGlu-HMRG activation in vitro and ex vivo in GGT positive cancer cells. Sensitivity and specificity of gGlu-HMRG and the Artemis on frozen tissue samples was 80% and 87%, respectively. Seven patients undergoing surgery for suspected oral cancer were included. In three patients fluorescence was observed at the resection margin. Those margins were either tumour-positive or within 1 mm of tumour. The margins of the other patients were clear (≥8 mm). Conclusion This study demonstrates the feasibility to detect tumour-positive resection margins with gGlu-HMRG and a clinical fluorescence imaging system. Applying this technique would enable intraoperative screening of the entire resection margin and allow direct re-resection in case of tumour-positivity.

Pre- and intraoperative diagnostic techniques facilitating tumor staging are of paramount importance in colorectal cancer surgery. The urokinase receptor (uPAR) plays an important role in the development of cancer, tumor invasion, angiogenesis, and metastasis and over-expression is found in the majority of carcinomas. This study aims to develop the first clinically relevant anti-uPAR antibody-based imaging agent that combines nuclear (111In) and real-time near-infrared (NIR) fluorescent imaging (ZW800-1). Conjugation and binding capacities were investigated and validated in vitro using spectrophotometry and cell-based assays. In vivo, three human colorectal xenograft models were used including an orthotopic peritoneal carcinomatosis model to image small tumors. Nuclear and NIR fluorescent signals showed clear tumor delineation between 24h and 72h post-injection, with highest tumor-to-background ratios of 5.0 ± 1.3 at 72h using fluorescence and 4.2 ± 0.1 at 24h with radioactivity. 1-2 mm sized tumors could be clearly recognized by their fluorescent rim. This study showed the feasibility of an uPAR-recognizing multimodal agent to visualize tumors during image-guided resections using NIR fluorescence, whereas its nuclear component assisted in the pre-operative non-invasive recognition of tumors using SPECT imaging. This strategy can assist in surgical planning and subsequent precision surgery to reduce the number of incomplete resections.

Background In oral cancer surgery, intraoperative optical imaging could help the surgeon to determine adequate tumor-free margins. Methods Tumor-specific near-infrared fluorescence agents targeting epidermal growth factor receptor (CW800 EGF) or glucose transporter system (CW800 2-DG) were administered to mice with tongue carcinoma and cervical lymph node metastases. Tumor growth was followed by bioluminescence imaging. Fluorescence signals were compared with a control group of healthy animals. Results Significantly higher fluorescence was found in tongue tumors and cervical lymph node metastases compared with that in control animals. Fluorescence correlated with histopathology. Tumor-to-background ratio of CW800 EGF in the tongue was 13.8 (SD = 6.1) and in the lymph nodes 15.7 (SD = 8.8). For CW800 2-DG, the tumor-to-background ratio in the tongue was 4.6 (SD = 2.1) and in the lymph nodes 33.9 (SD = 18.4). Conclusions Optical imaging can be used to detect oral cancer and cervical lymph node metastases and could potentially improve complete surgical resection by real-time image-guided surgery. © 2011 Wiley Periodicals, Inc. Head Neck, 2012

Background and Objectives Near-infrared (NIR) fluorescence optical imaging is a promising technique to assess the tumor margins during cancer surgery. This technique requires targeting by specific fluorescence agents to differentiate tumor from normal surrounding tissue. We assessed the feasibility of cancer detection using NIR fluorescence agents that target either αvβ3 integrins or the enhanced permeability and retention (EPR) effect in an orthotopic mouse model of oral cancer. Methods Binding of the integrin-targeted agent to tumor cells was assessed in vitro. Oral cancer was induced in 6 BALB/c nu/nu mice by submucosal inoculation of human OSC19-luc cells into the tongue. Tumor growth was followed with bioluminescence imaging. A combination of agents targeting integrins or EPR effect was injected followed by fluorescence imaging in vivo and ex vivo after resection of the tongues. Results Oral cancer was clearly demarcated in vitro; in vivo; and on histological analysis with sufficient tumor-to-background ratios of the contrast agents. Conclusion This study demonstrates the feasibility of optical imaging of oral squamous cell carcinoma based on targeting of αvβ3 integrins and the EPR effect. Once these NIR fluorescence agents become available for clinical testing, optical image-guided surgery could reduce residual disease after oral cancer surgery. J. Surg. Oncol. 2012; 105:714–718. © 2011 Wiley Periodicals, Inc.

In cancer surgery, intra-operative assessment of the tumor-free margin, which is critical for the prognosis of the patient, relies on the visual appearance and palpation of the tumor. Optical imaging techniques provide real-time visualization of the tumor, warranting intra-operative image-guided surgery. Within this field, imaging in the near-infrared light spectrum offers two essential advantages: increased tissue penetration of light and an increased signal-to-background-ratio of contrast agents. In this article, we review the various techniques, contrast agents, and camera systems that are currently used for image-guided surgery. Furthermore, we provide an overview of the wide range of molecular contrast agents targeting specific hallmarks of cancer and we describe perspectives on its future use in cancer surgery.

Quantification of tumor necrosis in cancer patients is of diagnostic value as the\ud amount of necrosis is correlated with disease prognosis and it could also be used to\ud predict early efficacy of anti-cancer treatments. In the present study, we identified\ud two near infrared fluorescent (NIRF) carboxylated cyanines, HQ5 and IRDye 800CW\ud (800CW), which possess strong necrosis avidity. In vitro studies showed that both\ud dyes selectively bind to cytoplasmic proteins of dead cells that have lost membrane\ud integrity. Affinity for cytoplasmic proteins was confirmed using quantitative structure\ud activity relations modeling. In vivo results, using NIRF and optoacoustic imaging,\ud confirmed the necrosis avid properties of HQ5 and 800CW in a mouse 4T1 breast\ud cancer tumor model of spontaneous necrosis. Finally, in a mouse EL4 lymphoma tumor\ud model, already 24 h post chemotherapy, a significant increase in 800CW fluorescence\ud intensity was observed in treated compared to untreated tumors. In conclusion, we\ud show, for the first time, that the NIRF carboxylated cyanines HQ5 and 800CW possess\ud strong necrosis avid properties in vitro and in vivo. When translated to the clinic,\ud these dyes may be used for diagnostic or prognostic purposes and for monitoring\ud in vivo tumor response early after the start of treatment.

Cancer patients could benefit from a surgical procedure that helps the surgeon to determine adequate tumor resection margins. Systemic injection of tumor-specific fluorescence agents with subsequent intraoperative optical imaging can guide the surgeon in this process. However, tumor heterogeneity hampers tumor-specific targeting. In addition, determination of adequate resection margins can be very challenging due to invasive tumor strands that are difficult to resolve and because of the confounding effect of variations in tissue optical properties in the surgical margin. We provide an overview of the "classic approach" of imaging tumor-specific targets or tumor-associated pathophysiological processes, and explain the limitations of these targeting strategies. It is proposed that problems of tumor heterogeneity can theoretically be circumvented by shifting focus of tumor targeting towards the follicle-stimulating hormone receptor (FSHR). Furthermore, we discuss why objective determination of resection margins is required to improve resection of the invasive strands, a goal that may be achieved by targeting the FSHR. When invasive strands would nevertheless extend beyond such a standardized resection margin, we suggest that adjuvant photodynamic therapy would be a very suitable therapeutic regimen. Finally, we describe how point optical spectroscopy can be used to scrutinize suspect tissue that is difficult to differentiate from normal tissue by measuring the local tissue optical properties to recover a local intrinsic fluorescence measurement.

Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photo-sensitizers chlorin e6 and Bremachlorin at t = [3; 4.5; 6; 24; 48] h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy. (C) 2014 Society of Photo-Optical Instrumentation Engineers (SPIE)

The effect of photodynamic therapy (PDT) is dependent on the localization of photosensitizer in the treatment volume at the time of illumination. Investigation of photosensitizer pharmacokinetics in and around the treatment volume aids in determining the optimal drug light interval for PDT.In this paper we have investigated the distribution of the photosensitizers chlorin e6 and Bremachlorin in the oral squamous cell carcinoma cell-line OSC19-Luc-Gfp in a tongue tumor, tumor boundary, invasive tumor boundary, and normal tongue tissue by the use of confocal microscopy of frozen sections. Tongues were harvested at t = [3, 4.5, 6, 24, 48] hours after injection.Both photosensitizers showed a decreasing fluorescence with increasing incubation time, and at all time points higher fluorescence was measured in tumor boundary than in tumor itself. For short incubation times, a higher fluorescence intensity was observed in the invasive tumor border and normal tissue compared to tumor tissue. Bremachlorin showed a small increase in tumor to normal ratio at 24 and 48 hours incubation time. Ce6 was undetectable at 48 hours. We did not find a correlation between photosensitizer localization and the presence of vasculature.The modest tumor/tumor boundary to normal selectivity of between 1.2 and 2.5 exhibited by Bremachlorin 24 and 48 hours after administration may allow selective targeting of tongue tumors. Further studies investigating the relationship between Bremachlorin concentration and therapeutic efficacy PDT with long incubation times are warranted.

Optical image-guided cancer surgery is a promising technique to adequately determine tumor margins by tumor-specific targeting, potentially resulting in complete resection of tumor tissue with improved survival. However, identification of the photons coming from the fluorescent contrast agent is complicated by autofluorescence, optical tissue properties, and accurate fluorescent targeting agents and imaging systems. All these factors have an important influence on the image that is presented to the surgeon. Considering the clinical consequences at stake, it is a prerequisite to answer the questions that are essential for the surgeon. What is optical image-guided surgery and how can it improve patient care? What should the oncologic surgeon know about the fundamental principles of optical imaging to understand which conclusions can be drawn from the images? And how do the limitations influence clinical decision making? This article discusses these questions and provides a clear overview of the basic principles and practical applications. Although there are limitations to the intrinsic capacity of the technique, when practical and technical surgical possibilities are considered, optical imaging can be a very powerful intraoperative tool in guiding the future oncologic surgeon toward radical resection and optimal clinical results. Clin Cancer Res; 19(14); 3745–54. ©2013 AACR.

Optical imaging is a promising technique to visualize cancer tissue during surgery. In this study, we explored the use of combinations of near-infrared (NIR) fluorescence agents that emit fluorescence signal at different wavelengths and each target specific tumor characteristics. Two combinations of agents (ProSense680 combined with 2DG CW800 and MMPSense680 combined with EGF CW800) were used to detect hypopharyngeal cancer in an animal model. ProSense680 and MMPSense680 detect increased activity of cathepsins and matrix metalloproteinases, respectively. These enzymes are mainly found in the invasive tumor border due to degradation of the extracellular matrix. 2DG CW800 detects tumor cells with high glucose metabolism and EGF CW800 is internalized by the epidermal growth factor receptor of tumor cells. Whole-body imaging revealed clear demarcation of tumor tissue using all four agents. The tumor-to-background ratio (standard deviation, p-value) was 3.69 (0.72, p < 0.001) for ProSense680; 4.26 (1.33, p < 0.001) for MMPSense680; 5.81 (3.59, p = 0.02) for 2DG CW800 and 4.84 (1.56, p < 0.001) for EGF CW800. Fluorescence signal corresponded with histopathology and immunohistochemistry, demonstrating signal of ProSense680 and MMPSense680 in the invasive tumor border, and signal of 2DG CW800 and EGF CW800 in the tumor tissue. In conclusion, we demonstrated the feasibility of dual wavelength tumor detection using different targeting strategies simultaneously in an animal model. Combined targeting at different wavelengths allowed simultaneous imaging of different tumor characteristics. NIR fluorescence optical imaging has the potential to be translated into the clinic in order to improve the complete removal of tumors by real-time image-guided surgery.

Objective To assess the feasibility of optical imaging using activatable near-infrared fluorescence (NIRF) agents to detect oral cancer and cervical lymph node metastasis in vivo. Design In vivo study. Setting University medical center. Subjects Female nude mice aged 4 to 6 weeks. Intervention Luciferase-expressing OSC-19-luc cells were injected into the tongues of nude mice. A control group of nude mice was injected in the tongue with a physiologic saline solution. Tumor growth was followed by bioluminescence imaging. After 3 weeks, animals were randomly allocated to intravenous administration of 1 of 2 activatable NIRF agents: ProSense680 or MMPSense680. Fluorescence imaging of the mice was performed, and the tumor to background ratio (TBR) was determined on histologic sections of the tongue and cervical lymph nodes after resection at necropsy. Main Outcome Measure Fluorescence signals. Results The fluorescence signals in tongue tumor and cervical lymph node metastases were significantly higher than those in control animals. The mean (SD) TBR of ProSense680 in the tongue was 15.8 (8.1) and in the lymph nodes was 11.8 (3.6). For MMPSense680, the mean (SD) TBR in the tongue was 18.6 (9.4) and in the lymph nodes was 10.5 (4.0). Conclusions Oral cancer and cervical lymph node metastases can be detected by targeting increased proteolytic activity at the tumor borders using NIRF optical imaging. These NIRF agents could be used for real-time image-guided surgery, which has the potential to improve the complete surgical resection of oral cancer.