Near-infrared Fluorescence Optical Imaging Research Articles

Therapy monitoring of psoriatic arthritis patients with and without skin involvement via objective evaluation of near infrared fluorescence optical imaging: A pilot study

Abstract In this work, we investigate the possibility of automatically and objectively evaluate an initialized therapy for psoriatic arthritis patients (therapy monitoring). Near infrared fluorescence optical imaging (NIR-FOI) is used to visualise the microcirculation in the hands, giving insights into possible inflammations (e.g. synovitis). Due to the time consuming and expansive data acquisition, we firstly investigate the feasibility of NIR-FOI in this scope of application using retrospective data. To increase the data quality and harmonize the heterogeneous patient population, certain inclusion criteria must be met, leaving only a limited amount of data sets for data analysis. A neural network was trained to segment the hands into areas of interest (e.g. joints and fingers) and an image registration pipeline was developed to compare identified regions of interest among different patient visits. It is shown that data from different visits are not directly comparable and thus, needs to be normalised. Due to the limited amount of meta data, only a simple normalisation can be performed. Even though with this normalisation the therapy monitoring cannot be carried out, the normalised data shows an increased agreement to a medical label in comparison to unnormalised data. Therefore, we conclude that further research can lead to a robust therapy monitoring algorithm.

Combining seeded region growing and k-nearest neighbours for the segmentation of routinely acquired spatio-temporal image data

PurposeThe acquisition conditions of medical imaging are often precisely defined, leading to a high homogeneity among different data sets. Nonetheless, outliers or artefacts still appear and need to be reliably detected to ensure a reliable diagnosis. Thus, the algorithms need to handle small sample sizes especially, when working with domain specific imaging modalities.MethodsIn this work, we suggest a pipeline for the detection and segmentation of light pollution in near-infrared fluorescence optical imaging (NIR-FOI), based on a small sample size. NIR-FOI produces spatio-temporal data with two spatial and one temporal dimension. To calculate a two-dimensional light pollution map for the entire image stack, we combine region growing and k-nearest neighbours (kNN), which classifies pixels into fore- and background by its entire temporal component. Thus, decision-making on reduced data is omitted.ResultsWe achieved a F_1 score of 0.99 for classifying a data set as light polluted or pollution-free. Additionally, we reached a total F_1 score of 0.90 for detecting regions of interest within the polluted data sets. Finally, an average Dice’s coefficient measuring the segmentation performance over all polluted data sets of 0.80 was accomplished.ConclusionsA Dice’s coefficient of 0.80 for the area segmentation does not seem perfect. However, there are two main factors, besides true prediction errors, lowering the score: Segmentation mistakes on small areas lead to a rapid decrease in the score and labelling errors due to complex data. However, in combination with the light-polluted data set and pollution area detection, these results can be considered successful and play a key role in our general goal: Exploiting NIR-FOI for the early detection of arthritis within hand joints.

Mediated Imaging and Improved Targeting of Farnesylthiosalicylic Acid Delivery for Pancreatic Cancer via Conjugation with Near-Infrared Fluorescence Heptamethine Carbocyanine Dye.

S-trans-trans-Farnesylthiosalicylic acid (FTS) is a Ras inhibitor that exhibits desirable anticancer property and currently undergoing clinical trials for pancreatic cancer (PC). However, its poor water solubility and low bioavailability have severely hampered clinical applications. A strategy to improve FTS bioavailability is to develop a suitable drug delivery method. Here, we use a near-infrared fluorescence (NIRF) heptamethine carbocyanine (HC) dye conjugated with FTS (to produce FTS-148) as a drug delivery system to enhance FTS bioavailability. We further investigate its tumor-targeting functions. FTS-148 displayed better bioavailability and photophysical property and selective recognition of cancer cells. FTS-148 significantly reduced PC cell proliferation, and more effective than FTS in restricting tumor growth both in a cell-derived xenograft (CDX) model and a patient-derived tumor xenograft (PDX) model. FTS-148 can specifically recognize PC cells in mice subcutaneous models or rabbit orthotopic models and allows real-time monitoring of the therapeutic effects by NIRF optical imaging. FTS-148 treatment significantly reduced Ras expression in PC cells and increased tumor tissue apoptosis. In short, FTS conjugated with HC dye had enhanced bioavailability and tumor-targeting property. It provides a potential agent for imaging and therapy of PC.

Optical Imaging of Triple-Negative Breast Cancer Cells in Xenograft Athymic Mice Using an ICAM-1-Targeting Small-Molecule Probe.

The development of early, accurate diagnostic strategies for triple-negative breast cancer (TNBC) remains a significant challenge. Intercellular adhesion molecule-1 (ICAM-1) overexpressed in human TNBC cells is a potential molecular target and biomarker for diagnosis. In this study, small-molecule probe (denoted as γ3-Cy5.5) constructed with a short 17-mer linear peptide (γ3) and near-infrared fluorescence (NIRF) dye cyanine 5.5 (Cy5.5) was used to detect the expression of ICAM-1 in vitro and in vivo, and to diagnose TNBC via NIRF imaging. Western blotting and flow cytometric analysis were used for the detection of ICAM-1 expression in MDA-MB-231 and MCF-7 cells. The cytotoxicity of the small-molecule probe γ3-Cy5.5 was detected using the CCK8 assay. The in vitro targeting of the small-molecule probe γ3-Cy5.5 was verified via flow cytometry and a laser scanning confocal microscope. Finally, the targeting of small-molecule probe in vivo and ex vivo was observed by NIRF imaging. Western blotting and flow cytometry demonstrate that ICAM-1 was highly expressed in the MDA-MB-231 TNBC cell line. Laser confocal microscopy and flow cytometry results show that TNBC cells have an increased cellular uptake of γ3-Cy5.5 compared to the control probe γ3S-Cy5.5. With in vivo NIRF, a significantly higher Cy5.5 signal appeared in the tumors of mice administered γ3-Cy5.5 than those treated with γ3S-Cy5.5. The target-to-background ratio observed on the NIRF images was significantly higher in the γ3-Cy5.5 group (10.2, 13.6) compared with the γ3S-Cy5.5 group (4.4, 4.0) at 1 and 2h, respectively. This is the first report of the use of ICAM-1-specific small-molecule probe for in vivo NIRF optical imaging of TNBC. This method provides a noninvasive and specific strategy for the early diagnosis of TNBC.

Doxorubicin-loaded protease-activated near-infrared fluorescent polymeric nanoparticles for imaging and therapy of cancer.

IntroductionDespite significant progress in the field of oncology, cancer remains one of the leading causes of death. Chemotherapy is one of the most common treatment options for cancer patients but is well known to result in off-target toxicity. Theranostic nanomedicines that integrate diagnostic and therapeutic functions within an all-in-one platform can increase tumor selectivity for more effective chemotherapy and aid in diagnosis and monitoring of therapeutic responses.Material and methodsIn this work, theranostic nanoparticles were synthesized with commonly used biocompatible and biodegradable polymers and used as cancer contrast and therapeutic agents for optical imaging and treatment of breast cancer. These core–shell nanoparticles were prepared by nanoprecipitation of blends of the biodegradable and biocompatible amphiphilic copolymers poly(lactic-co-glycolic acid)-b-poly-l-lysine and poly(lactic acid)-b-poly(ethylene glycol). Poly-l-lysine in the first copolymer was covalently decorated with near-infrared fluorescent Alexa Fluor 750 molecules.ResultsThe spherical nanoparticles had an average size of 60–80 nm. The chemotherapeutic drug doxorubicin was encapsulated in the core of nanoparticles at a loading of 3% (w:w) and controllably released over a period of 30 days. A 33-fold increase in near-infrared fluorescence, mediated by protease-mediated cleavage of the Alexa Fluor 750-labeled poly-l-lysine on the surface of the nanoparticles, was observed upon interaction with the model protease trypsin. The cytocompatibility of drug-free nanoparticles and growth inhibition of drug-loaded nanoparticles on MDA-MB-231 breast cancer cells were investigated with a luminescence cell-viability assay. Drug-free nanoparticles were found to cause minimal toxicity, even at high concentrations (0.2–2,000 µg/mL), while doxorubicin-loaded nanoparticles significantly reduced cell viability at drug concentrations >10 µM. Finally, the interaction of the nanoparticles with breast cancer cells was studied utilizing fluorescence microscopy, demonstrating the potential of the nanoparticles to act as near-infrared fluorescence optical imaging agents and drug-delivery carriers.ConclusionDoxorubicin-loaded, enzymatically activatable nanoparticles of less than 100 nm were prepared successfully by nanoprecipitation of copolymer blends. These nanoparticles were found to be suitable as controlled drug delivery systems and contrast agents for imaging of cancer cells.

Near-infrared fluorescence optical imaging demonstrates that C5a-C5aR1 signaling impairs normal lymphatic function

Near-infrared fluorescence optical imaging demonstrates that C5a-C5aR1 signaling impairs normal lymphatic function

In Vivo Tumor Angiogenesis Imaging Using Peptide-Based Near-Infrared Fluorescent Probes.

Near-infrared fluorescence (NIRF) imaging is an emerging imaging technique for studying diseases at the molecular level. Optical imaging with a near-infrared emitting fluorophore for targeting tumor angiogenesis offers a noninvasive method for early tumor detection and efficient monitoring of tumor response to anti-angiogenesis therapy. CD13 receptor, a zinc-dependent membrane-bound ectopeptidase, plays important roles in regulating tumor angiogenesis and the growth of new blood vessels. In this chapter, we use CD13 receptor as an example to demonstrate how to construct CD13-specific NGR-containing peptides via bioorthogonal click chemistry for visualizing and quantifying the CD13 receptor expression in vivo by means of NIRF optical imaging.

Site-specifically labeled CA19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer

Molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF)-guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of pancreatic cancer. PET and NIRF optical imaging offer complementary clinical applications, enabling the noninvasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. We report the development of PET, NIRF, and dual-modal (PET/NIRF) imaging agents, using 5B1, a fully human monoclonal antibody that targets CA19.9, a well-established pancreatic cancer biomarker. Desferrioxamine (DFO) and/or a NIRF dye (FL) were conjugated to the heavy-chain glycans of 5B1, using a robust and reproducible site-specific (ss) labeling methodology to generate three constructs ((ss)DFO-5B1, (ss)FL-5B1, and (ss)dual-5B1) in which the immunoreactivity was not affected by the conjugation of either label. Each construct was evaluated in a s.c. xenograft model, using CA19.9-positive (BxPC3) and -negative (MIAPaCa-2) human pancreatic cancer cell lines. Each construct showed exceptional uptake and contrast in antigen-positive tumors with negligible nonspecific uptake in antigen-negative tumors. Additionally, the dual-modal construct was evaluated in an orthotopic murine pancreatic cancer model, using the human pancreatic cancer cell line, Suit-2. The (ss)dual-5B1 demonstrated a remarkable capacity to delineate metastases and to map the sentinel lymph nodes via tandem PET-computed tomography (PET/CT) and NIRF imaging. Fluorescence microscopy, histopathology, and autoradiography were performed on representative sections of excised tumors to visualize the distribution of the constructs within the tumors. These imaging tools have tremendous potential for further preclinical research and for clinical translation.

Non-invasive imaging and cellular tracking of pulmonary emboli by near-infrared fluorescence and positron-emission tomography.

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements.

Multifunctional calcium phosphate nanoparticles for combining near-infrared fluorescence imaging and photodynamic therapy

Photodynamic therapy (PDT) of tumors causes skin photosensitivity as a result of unspecific accumulation behavior of the photosensitizers. PDT of tumors was improved by calcium phosphate nanoparticles conjugated with (i) Temoporfin as a photosensitizer, (ii) the RGDfK peptide for favored tumor targeting and (iii) the fluorescent dye molecule DY682-NHS for enabling near-infrared fluorescence (NIRF) optical imaging in vivo. The nanoparticles were characterized with regard to size, spectroscopic properties and uptake into CAL-27 cells. The nanoparticles had a hydrodynamic diameter of approximately 200nm and a zeta potential of around +22mV. Their biodistribution at 24h after injection was investigated via NIRF optical imaging. After treating tumor-bearing CAL-27 mice with nanoparticle-PDT, the therapeutic efficacy was assessed by a fluorescent DY-734-annexin V probe at 2days and 2weeks after treatment to detect apoptosis. Additionally, the contrast agent IRDye® 800CW RGD was used to assess tumor vascularization (up to 4weeks after PDT). After nanoparticle-PDT in mice, apoptosis in the tumor was detected after 2days. Decreases in tumor vascularization and tumor volume were detected in the next few days. Calcium phosphate nanoparticles can be used as multifunctional tools for NIRF optical imaging, PDT and tumor targeting as they exhibited a high therapeutic efficacy, being capable of inducing apoptosis and destroying tumor vascularization.

In vivo dual-modality MR and near-infrared fluorescence imaging of gastric cancer neovasculature with targeted multi-functionalized nanoparticles

Objective: To explore the feasibility of dual-modality probe to in vivo target angiogenesis of gastric cancer on MRI and near-infrared fluorescence (NIRF) optical imaging. Methods: Cy5.5-GX1 with magnetic FeO nanoparticles were conjugated, resulting in dual-modality probe DPs. The hydrodynamic size and Zeta potential of DPs were analyzed by nano-ZS. Twelve male nude mice were obtained and established gastric adenocarcinoma subcutaneous transplantation tumor model. Twelve nude mice were divided into experimental group and control group (n=6 for each) by using completely random experimental design, then intravenously injected the same volume of DPs and FeO-Cy5.5. MRI was applied before and 4, 8, 12, 18, 24 hours respectively after injections of nanoparticles to follow the changes of signals. For the evaluation of the MR images, the contrast to noise ratio (CNR) were calculated, and the different groups were compared with analysis of variance. MR images of the same level were selected before and 12 h after injections of nanoparticles in two groups and the percentage of reduced pixels was calculated, which were compared with the paired t test. At the same time, the time-resolved accumulation of the nanoparticles in the tumors were observed by using in vivo fluorescence imaging at 0, 4, 8, 12, 18, 24 hours after injecting nanoparticles, respectively. Then tumor and normal tissues were collected, and ex vivo optical imaging and histopathology were performed on the tissues. Results: At room temperature, the hydrodynamic size of FeO-Cy5.5 was(38.23 ± 0.06)nm and the Zeta potential was (0.29 ± 0.16)mV, and the hydrodynamic size of DPs was (39.49 ± 0.16) nm and the Zeta potential was (- 4.15 ± 0.79) mV. The coupled rate of DPs with polypeptide was >90%. The MRI signals of reticuloendothelial system were reduced significantly in experiment and control group. The tumor CNR of experiment group before and 4, 8, 12, 18, 24 h after injection were 18.27 ± 2.19, 18.40 ± 2.00, 10.22 ± 1.97, 9.25 ± 0.44, 20.28 ± 1.46 and 22.41 ± 1.71 (F= 49.55, P 0.05). The percentage of MR signals decrease of the tumor periphery and central area were(33.0 ± 2.7)% and (22.0 ± 1.6)%, respectively in experiment group and that of control group were (8.3 ± 1.2)% and (3.8 ± 0.9)% respectively (t values were - 7.872 and 6.678, P< 0.01). The tumor fluorescence intensity of experimental group showed the high contrast to background tissue during 2 to 48 h p.i, but there was no significant difference in control group. Furthermore, ex vivo evaluation of excised organs showed DPs was predominantly taken up by the lumor and lung at 48 h p.i. Tumor neovascularization was mainly distributed in the tumor periphery area. Iron-positive cells were detected in the tumor of experiment group. The normal kidney tissues of the two groups and the tumor of control group were consistently negative for iron staining. Conclusion: DPs can selectively be delivered into gastric cancer and this novel receptor-targeted nanoparlicle could be used for MRI and NIRF optical imaging.

Intraoperative assessment of margins in breast conservative surgery--still in use?

A positive margin in breast conserving surgery is associated with an increased risk of local recurrence. Failure to achieve clear margins results in re-excision procedures. Methods for intraoperative assessment of margins have been developed, such as frozen section analysis, touch preparation cytology, near-infrared fluorescence optical imaging, x-ray diffraction technology, high-frequency ultrasound, micro-CT, and radiofrequency spectroscopy. In this article, options that might become the method of choice in the future are discussed.

Abstract P2-18-14: MarginProbe® - Final results of the German post-market study in breast conserving surgery of ductal carcinoma in situ

Abstract Background: Surgical margins in breast conserving surgery (BCS) of ductal carcinoma in situ (DCIS) as well as invasive cancer are a subject of intense discussion. A positive margin is still one of the strongest predictor for risk of local recurrence of intraductal and invasive breast cancer. Intraoperative margin assessment of DCIS is difficult and existing options are unsatisfying. Aim of our study was to evaluate the performance of MarginProbe®(Dune Medical Devices, Boston, MA, USA) in assessing surgical margins for DCIS and the contribution to lowering the re-excision rate after initial BCS. Material and methods: The device includes a disposable hand-held probe and a console, is based on radiofrequency spectroscopy, and detects differences in dielectric properties between normal and malignant breast tissue. The multicentre single arm, post market study was performed on 55 patients at 3 German sites under approval of institutional review boards. MarginProbe® was used as an adjunctive tool to the current practice. All specimens were sent for paraffin embedded pathological analysis. The procedure success was defined as both: negative margins after initial BCS; and early identification of an extended lesion, with conversion to mastectomy instead of performing a re-excision BCS. Results: From September 2009 - May 2010, 42/55 patients were eligible for this analysis. In comparison with a historical re-excision rate of 39% for DCIS patients undergoing BCS, use of MarginProbe® led to a reduction of the re-excision rate by 56%, down to 17% (p = 0.018) (surgical margin width of 5 mm). The procedure success was also significantly improved by more than 3-fold (p&amp;lt;0.0001), from 14% (6/42) by standard of care (SOC) alone, to 57% (24/42) with use of the device. In 21% (9/42) of the cases use of the device led to a direct conversion to mastectomy due to extensive disease identified, sparing an additional re-excision BCS. Resected average main specimen tissue volume was 34.6 cc. Tissue volume associated with false positive margins was, on average, 7.5 cc per patient. Cosmetic outcomes were not different. Discussion: In synopsis with the other two clinical trials (MAST and US pivotal study) we found our data consistent. Each trial reported a reduction of the re-resection rate by more than 50%. Our results demonstrate that adjunctive use of the device is effective in achieving procedure success and reducing re-excisions for DCIS patients. Several options for intraoperative margin assessment exist, but for DCIS they are time-consuming and insufficient (frozen section, touch prep cytology) or experimental (near-infrared fluorescence optical imaging, high-frequency ultrasound). Usually, the pathologist provides final analysis of the lesion extension and margin assessment several days after operation. Conclusion: MarginProbe® is a device that provides a fast and effective technique for intraoperative margin assessment that is already used in daily routine. It lowers the re-excision rate for both DCIS and invasive cancer significantly, by &amp;gt;50%, without any influence on patient's cosmesis. It may allow the surgeon to perform oncoplastic or reconstructive breast surgery and IORT more safely in the future. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-18-14.

The future for surgical margins

The future for surgical margins

Assessment of Rat Antigen-Induced Arthritis and Its Suppression During Glucocorticoid Therapy by Use of Hemicyanine Dye Probes With Different Molecular Weight in Near-Infrared Fluorescence Optical Imaging

Arthritic joints are ideal disease entities to be assessed via optical imaging. Here, we investigated the selective accumulation behavior of two differently sized hemicyanine optical probes in arthritic joints and its modification during glucocorticoid therapy in the course of inflammation. The well-standardized preclinical antigen-induced arthritis (AIA) model in rats was used. The animals were divided into 3 groups: arthritic, arthritic and dexamethasone-treated, and immunized only. After intravenous coinjection of DY-752 (size, 800 Da) and DY-682-(rat) IgG (size, 150 kDa) probes, spectrally unmixed near-infrared fluorescence images were acquired and analyzed semiquantitatively. Probe organ distribution, joint swelling, blood cell counts, joint vessel density, and histological scoring of arthritis were determined. The local joint accumulation kinetics of the DY-752 probe differed from the DY-682-IgG one. In the course of AIA, probe signaling in arthritic joints was similar between each other. Joint swelling and histological scoring were in accordance with signaling. Dexamethasone treatment of rats with AIA significantly reduced both the near-infrared fluorescence signals and severity of arthritis but did not change the joint vascular density or the uptake of the probes by phagocytes. A differential biodistribution of both probes was encountered, but such an accumulation was prevented by dexamethasone treatment. Near-infrared fluorescence signaling in the course of AIA closely reflects the pathophysiological events of the arthritic joint and the effects of therapy independently of the molecular size of the probe. The results show the suitability of our hemicyanine probes for imaging of arthritis.

Non-invasive imaging for studying anti-angiogenic therapy effects

Noninvasive imaging plays an emerging role in preclinical and clinical cancer research and has high potential to improve clinical translation of new drugs. This article summarises and discusses tools and methods to image tumour angiogenesis and monitor anti-angiogenic therapy effects. In this context, micro-computed tomography (µCT) is recommended to visualise and quantify the micro-architecture of functional tumour vessels. Contrast-enhanced ultrasound (US) and magnetic resonance imaging (MRI) are favourable tools to assess functional vascular parameters, such as perfusion and relative blood volume. These functional parameters have been shown to indicate anti-angiogenic therapy response at an early stage, before changes in tumour size appear. For tumour characterisation, the imaging of the molecular characteristics of tumour blood vessels, such as receptor expression, might have an even higher diagnostic potential and has been shown to be highly suitable for therapy monitoring as well. In this context, US using targeted microbubbles is currently evaluated in clinical trials as an important tool for the molecular characterisation of the angiogenic endothelium. Other modalities, being preferably used for molecular imaging of vessels and their surrounding stroma, are photoacoustic imaging (PAI), near-infrared fluorescence optical imaging (OI), MRI, positron emission tomography (PET) and single photon emission computed tomography (SPECT). The latter two are particularly useful if very high sensitivity is needed, and/or if the molecular target is difficult to access. Carefully considering the pros and cons of different imaging modalities in a multimodal imaging setup enables a comprehensive longitudinal assessment of the (micro)morphology, function and molecular regulation of tumour vessels.

Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

ObjectiveBone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.MethodsThe paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.ResultsConjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.ConclusionsThis preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.

Dual wavelength tumor targeting for detection of hypopharyngeal cancer using near-infrared optical imaging in an animal model

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.

Detection of Oral Squamous Cell Carcinoma and Cervical Lymph Node Metastasis Using Activatable Near-Infrared Fluorescence Agents

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. In vivo study. University medical center. Female nude mice aged 4 to 6 weeks. 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. Fluorescence signals. 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). 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.

Optical Imaging of CCK2/Gastrin Receptor-Positive Tumors With a Minigastrin Near-Infrared Probe

A variety of tumors in different organs with good accessibility to near-infrared light express the cholecystokinin-2 (CCK₂)/gastrin receptor. Therefore, the applicability of fluorescence optical imaging was assessed using a novel peptide probe. This study was approved by the regional animal committee. Our optical peptide probe (DY-minigastrin) was synthesized by coupling a hemicyanine dye to a gastrin derivative peptide (minigastrin). In vitro CCK₂/gastrin receptor identification was performed in receptor-positive HT-29 and negative A-375 cells using flow cytometry, laser scanning microscopy, and macroscopic near-infrared fluorescent (NIRF) imaging. For in vivo studies, tumor cells were implanted into mice, and DY-minigastrin in presence or absence of nonlabeled minigastrin (control of signaling specificity) was applied intravenously. Fluorescence signals in tumors and organs were recorded and statistically analyzed. Flow cytometry, laser scanning microscopy, and in vitro macroscopic imaging of cell pellets revealed a distinct accumulation of our minigastrin probe in HT-29 cells, showing distinct probe internalization. In vivo NIRF whole-body animal imaging, again, demonstrated a clear depiction of HT-29 tumors, which was reversed by blocking with nonlabeled minigastrin. Semi-quantitative fluorescence analysis and histologic observations were in agreement with these observations. A distinct probe organ distribution was observed. Our observations indicate that DY-minigastrin-based NIRF optical imaging of CCK₂/gastrin receptor protein is feasible. Because of its widespread occurrence in different tumor types, endoscopic, laparoscopic, and tomographic receptor imaging could be accomplished in the near future.