Fluorescence-guided tumor detection with a novel anti-EpCAM targeted antibody fragment: Preclinical validation.

Tumor-specific fluorescent imaging agents are moving towards the clinic, supporting surgeons with real-time intraoperative feedback about tumor locations. The epithelial cell adhesion molecule (EpCAM) is considered as one of the most promising tumor-specific proteins due its high overexpression on epithelial-derived cancers. This study describes the development and evaluation of EpCAM-F800, a novel fluorescent anti-EpCAM antibody fragment, for intraoperative tumor imaging. Fab production, conjugation to the fluorophore IRDye 800CW, and binding capacities were determined and validated using HPLC, spectrophotometry and cell-based assays. In vivo, dose escalation-, blocking-, pharmacokinetic- and biodistribution studies (using both fluorescence and radioactivity) were performed, next to imaging of clinically relevant orthotopic xenografts for breast and colorectal cancer. EpCAM-F800 targets EpCAM with high specificity in vitro, which was validated using in vivo blocking experiments with a 10x higher dose of unlabeled Fab. The optimal dose range for fluorescence tumor detection in mice was 1-5 nmol (52-260 µg), which corresponds to a human equivalent dose of 0.2-0.8 mg/kg. Biodistribution showed high accumulation of EpCAM-F800 in tumors and metabolizing organs. Breast and colorectal tumors could clearly be visualized within 8 h post-injection and up to 96 h, while the agent already showed homogenous tumor distribution within 4 h. The blood half-life was 4.5 h. This study describes the development and evaluation of a novel EpCAM-targeting agent and the feasibility to visualize breast and colorectal tumors by fluorescence imaging during resections. EpCAM-F800 will be translated for clinical use, considering its abundance in a broad range of tumor types.

Biomarker expression in rectal cancer tissue before and after neoadjuvant therapy.

PURPOSE: Intraoperative identification of rectal cancer (RC) can be challenging, especially because of fibrosis after treatment with preoperative chemo- and radiotherapy (CRT). Tumor-targeted fluorescence imaging can enhance the contrast between tumor and normal tissue during surgery. Promising targets for RC imaging are carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM) and the tyrosine-kinase receptor Met (c-Met). The effect of CRT on their expression determines their applicability for imaging. Therefore, we investigated whether CRT modifies expression patterns in tumors, lymph node (LN) metastases and adjacent normal rectal tissues. PATIENTS AND METHODS: Preoperative biopsies, primary tumor specimens and metastatic LNs were collected from 38 RC patients who did not receive CRT (cohort 1) and 34 patients who did (cohort 2). CEA, EpCAM and c-Met expression was determined using immunohistochemical staining and was semiquantified by a total immunostaining score (TIS), consisting of the percentage and intensity of stained tumor cells (0-12). RESULTS: In both cohorts CEA, EpCAM and c-Met were significantly highly expressed in >60% of tumor tissues compared with adjacent normal epithelium (T/N ratio, P<0.01). EpCAM showed the most homogenous expression in tumors, whereas CEA showed the highest T/N ratio. Most importantly, CEA and EpCAM expression did not significantly change in normal or neoplastic RC tissue after CRT, whereas levels of c-Met changed (P=0.02). Tissues of eight patients with a pathological complete response after CRT showed expression of all biomarkers with TIS close to normal epithelium. CONCLUSION: Histological evaluation shows that CEA, EpCAM and c-Met are suitable targets for RC imaging, because all three are significantly enhanced in cancer tissue from primary tumors or LN metastases compared with normal adjacent tissue. Furthermore, the expression of CEA and EpCAM is not significantly changed after CRT. These data underscore the applicability of c-Met and especially, CEA and EpCAM as targets for image-guided RC surgery, both before and after CRT.

Detecting tumour-positive resection margins after oral cancer surgery by spraying a fluorescent tracer activated by gamma-glutamyltranspeptidase.

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.

Selection of optimal molecular targets for tumor-specific imaging in pancreatic ductal adenocarcinoma.

Discrimination of pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis (CP) or peritumoral inflammation is challenging, both at preoperative imaging and during surgery, but it is crucial for proper therapy selection. Tumor-specific molecular imaging aims to enhance this discrimination and to help select and stratify patients for resection. We evaluated various biomarkers for the specific identification of PDAC and associated lymph node metastases. Using immunohistochemistry (IHC), expression levels and patterns were investigated of integrin αvß6, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), Cathepsin E (Cath E), epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), thymocyte differentiation antigen 1 (Thy1), and urokinase-type plasminogen activator receptor (uPAR). In a first cohort, multiple types of pancreatic tissue were evaluated (n=62); normal pancreatic tissue (n=8), CP (n=7), PDAC (n=9), tumor associated lymph nodes (n=32), and PDAC after neoadjuvant radiochemotherapy (n=6). In a second cohort, tissues were investigated (n=55) with IHC and immunofluorescence (IF) for concordance of biomarker expression in all tissue types, obtained from an individual patient. Integrin αvß6 and CEACAM5 showed significantly higher expression levels in PDAC versus normal pancreatic tissue (P=0.001 and P<0.001, respectively) and CP (P=0.003 and P<0.001, respectively). Avß6 and CEACAM5 expression identified tumor-positive lymph nodes correctly in 84% and 68%, respectively, and in 100% of tumor-negative nodes for both biomarkers. In conclusion, αvß6 and CEACAM5 are excellent biomarkers to differentiate PDAC from surrounding tissue and to identify lymph node metastases. Individually or combined, these biomarkers are promising targets for tumor-specific molecular imaging of PDAC.

Prognostic Impact of Urokinase Plasminogen Activator Receptor Expression in Pancreatic Cancer: Malignant Versus Stromal Cells.

The urokinase plasminogen activator receptor (uPAR) has been proposed as a potential prognostic factor for various malignancies. The aim of this study is to assess the prognostic value of uPAR expression in neoplastic and stromal cells of patients with pancreatic adenocarcinoma. Urokinase plasminogen activator receptor expression was determined by immunohistochemistry in 122 pancreatic ductal adenocarcinomas. Kaplan-Meier and Cox regression analyses were used to determine the association with survival. Respectively 66%, 82% and 62% of patients with pancreatic cancer expressed uPAR in neoplastic cells, stromal, and in both combined. Multivariate analysis showed a significant inverse association between uPAR expression in both neoplastic and stromal cells and overall survival. The prognostic impact of uPAR in stromal cells is substantial, but not as pronounced as that of uPAR expression in neoplastic cells. This study suggests a role for uPAR as a biomarker to single out higher risk subgroups of patients with pancreatic cancer.

SGM-101: An innovative near-infrared dye-antibody conjugate that targets CEA for fluorescence-guided surgery.

PURPOSE: Fluorescence-guided surgery (FGS) provides surgeons with new opportunities to improve real-time cancer nodule detection and tumor margin visualization. Currently, the most important challenge in this field is the development of fluorescent dyes that specifically target tumors. We developed, characterized and evaluated SGM-101, an innovative antibody-dye conjugate in which the fluorochrome BM104, which has an absorbance band centered at 700 nm, is coupled to a chimeric monoclonal antibody (mAb) against carcinoembryonic antigen (CEA). METHODS: The dye to mAb ratio, binding to CEA and photobleaching of SGM-101 were determined. FGS was performed and results analyzed using different mouse models of human digestive tumors. RESULTS: SGM-101 allowed the detection of tumor nodules in three different colon cancer models: LS174T human colorectal adenocarcinoma cell-induced peritoneal carcinomatosis (PC) and liver metastases, and orthotopic grafts of HT29 human colorectal adenocarcinoma cells. In the PC model, submillimeter-sized nodules were detected during SGM-101-based FGS and SGM-101 predictive positive values ranged from 99.04% to 90.24% for tumor nodules >10 mg and nodules <1 mg, respectively. Similarly, in the orthotopic model of pancreatic cancer using BxPC3 (pancreas adenocarcinoma) cells, SGM-101 could clearly delineate tumors in vivo with a tumor-to-background ratio of 3.5, and penetrated in tumor nodules, as demonstrated by histological analysis. Free BM105 dye (BM104 with an activated ester for conjugation to the antibody) and an irrelevant conjugate did not induce any NIR fluorescence. CONCLUSION: These preclinical data indicate that SGM-101 is an attractive candidate for FGS of CEA-expressing tumors and is currently assessed in clinical trials.

Inhibition of 14q32 microRNA miR-495 reduces lesion formation, intimal hyperplasia and plasma cholesterol levels in experimental restenosis.

BACKGROUND AND AIMS: We aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated for coronary artery disease and peripheral arterial disease. We have previously shown that inhibition of 14q32 microRNAs leads to increased post-ischemic neovascularization, and microRNA miR-494 also decreased atherosclerosis, while increasing plaque stability. We hypothesized that 14q32 microRNA inhibition has beneficial effects on intimal hyperplasia, as well as accelerated atherosclerosis. METHODS: Non-constrictive cuffs were placed around both femoral arteries of C57BL/6J mice to induce intimal hyperplasia. Accelerated atherosclerotic plaque formation was induced in hypercholesterolemic ApoE-/- mice by placing semi-constrictive collars around both carotid arteries. 14q32 microRNAs miR-329, miR-494 and miR-495 were inhibited in vivo using Gene Silencing Oligonucleotides (GSOs). RESULTS: GSO-495 administration led to a 32% reduction of intimal hyperplasia. Moreover, the number of macrophages in the arterial wall of mice treated with GSO-495 was reduced by 55%. Inhibition of miR-329 and miR-494 had less profound effects on intimal hyperplasia. GSO-495 administration also decreased atherosclerotic plaque formation by 52% and plaques of GSO-495 treated animals showed a more stable phenotype. Finally, cholesterol levels were also decreased in GSO-495 treated animals, via reduction of the VLDL-fraction. CONCLUSIONS: GSO-495 administration decreased our primary outcomes, namely intimal hyperplasia, and accelerated atherosclerosis. GSO-495 administration also favourably affected multiple secondary outcomes, including macrophage influx, plaque stability and total plasma cholesterol levels. We conclude that 14q32 microRNA miR-495 is a promising target for prevention of restenosis.

Real-time near-infrared fluorescence imaging using cRGD-ZW800-1 for intraoperative visualization of multiple cancer types.

Incomplete resections and damage to critical structures increase morbidity and mortality of patients with cancer. Targeted intraoperative fluorescence imaging aids surgeons by providing real-time visualization of tumors and vital structures. This study evaluated the tumor-targeted zwitterionic near-infrared fluorescent peptide cRGD-ZW800-1 as tracer for intraoperative imaging of multiple cancer types. cRGD-ZW800-1 was validated in vitro on glioblastoma (U-87 MG) and colorectal (HT-29) cell lines. Subsequently, the tracer was tested in orthotopic mouse models with HT-29, breast (MCF-7), pancreatic (BxPC-3), and oral (OSC-19) tumors. Dose-ranging studies, including doses of 0.25, 1.0, 10, and 30 nmol, in xenograft tumor models suggest an optimal dose of 10 nmol, corresponding to a human equivalent dose of 63 µg/kg, and an optimal imaging window between 2 and 24 h post-injection. The mean half-life of cRGD-ZW800-1 in blood was 25 min. Biodistribution at 4 h showed the highest fluorescence signals in tumors and kidneys. In vitro and in vivo competition experiments showed significantly lower fluorescence signals when U-87 MG cells (minus 36%, p = 0.02) or HT-29 tumor bearing mice (TBR at 4 h 3.2 ± 0.5 vs 1.8 ± 0.4, p = 0.03) were simultaneously treated with unlabeled cRGD. cRGD-ZW800-1 visualized in vivo all colorectal, breast, pancreatic, and oral tumor xenografts in mice. Screening for off-target interactions, cRGD-ZW800-1 showed only inhibition of COX-2, likely due to binding of cRGD-ZW800-1 to integrin αVß3. Due to its recognition of various integrins, which are expressed on malignant and neoangiogenic cells, it is expected that cRGD-ZW800-1 will provide a sensitive and generic tool to visualize cancer during surgery.

Selecting Targets for Tumor Imaging: An Overview of Cancer-Associated Membrane Proteins.

Tumor targeting is a booming business: The global therapeutic monoclonal antibody market accounted for more than $78 billion in 2012 and is expanding exponentially. Tumors can be targeted with an extensive arsenal of monoclonal antibodies, ligand proteins, peptides, RNAs, and small molecules. In addition to therapeutic targeting, some of these compounds can also be applied for tumor visualization before or during surgery, after conjugation with radionuclides and/or near-infrared fluorescent dyes. The majority of these tumor-targeting compounds are directed against cell membrane-bound proteins. Various categories of targetable membrane-bound proteins, such as anchoring proteins, receptors, enzymes, and transporter proteins, exist. The functions and biological characteristics of these proteins determine their location and distribution on the cell membrane, making them more, or less, accessible, and therefore, it is important to understand these features. In this review, we evaluate the characteristics of cancer-associated membrane proteins and discuss their overall usability for cancer targeting, especially focusing on imaging applications.

Near-infrared fluorescence sentinel lymph node detection in gastric cancer: A pilot study.

AIM: To investigate feasibility and accuracy of near-infrared fluorescence imaging using indocyanine green: nanocolloid for sentinel lymph node (SLN) detection in gastric cancer. METHODS: A prospective, single-institution, phase I feasibility trial was conducted. Patients suffering from gastric cancer and planned for gastrectomy were included. During surgery, a subserosal injection of 1.6 mL ICG:Nanocoll was administered around the tumor. NIR fluorescence imaging of the abdominal cavity was performed using the Mini-FLARE™ NIR fluorescence imaging system. Lymphatic pathways and SLNs were visualized. Of every detected SLN, the corresponding lymph node station, signal-to-background ratio and histopathological diagnosis was determined. Patients underwent standard-of-care gastrectomy. Detected SLNs outside the standard dissection planes were also resected and evaluated. RESULTS: Twenty-six patients were enrolled. Four patients were excluded because distant metastases were found during surgery or due to technical failure of the injection. In 21 of the remaining 22 patients, at least 1 SLN was detected by NIR Fluorescence imaging (mean 3.1 SLNs; range 1-6). In 8 of the 21 patients, tumor-positive LNs were found. Overall accuracy of the technique was 90% (70%-99%; 95%CI), which decreased by higher pT-stage (100%, 100%, 100%, 90%, 0% for respectively Tx, T1, T2, T3, T4 tumors). All NIR-negative SLNs were completely effaced by tumor. Mean fluorescence signal-to-background ratio of SLNs was 4.4 (range 1.4-19.8). In 8 of the 21 patients, SLNs outside the standard resection plane were identified, that contained malignant cells in 2 patients. CONCLUSION: This study shows successful use of ICG:Nanocoll as lymphatic tracer for SLN detection in gastric cancer. Moreover, tumor-containing LNs outside the standard dissection planes were identified.

Concordance of folate receptor-α expression between biopsy, primary tumor and metastasis in breast cancer and lung cancer patients.

Folate receptor alpha (FRα) is known to be upregulated in a variety of cancers, including non-small cell lung cancer (NSCLC) and breast cancer. To ensure reliable implementation of diagnostic- and therapeutic agents, concordance of FRα expression between biopsy, primary tumor and metastases is important. Using immunohistochemistry (Mab 26B3.F2) these concordances were investigated in 60 NSCLC and 40 breast cancer patients. False positivity of FRα expression on breast and lung cancer biopsies was limited to less than 5%. In NSCLC, FRα expression was shown in 21/34 adenocarcinomas and 4/26 squamous cell carcinomas (SCC). Concordance of FRα expression between biopsy and primary tumor was achieved in respectively 83% and 91% of adenocarcinomas and SCCs. Approximately 80% of all local and distant metastases of NSCLC patients showed concordant FRα expression as their corresponding primary tumor. In breast cancer, FRα positivity was shown in 12/40 biopsies, 20/40 lumpectomies and 6/20 LN metastases, with concordance of 68% between biopsy and primary tumor and 60% between primary tumor and LN metastases. In conclusion, this study shows high concordance rates of FRα expression between biopsies and metastases compared to primary NSCLC and breast cancers, underscoring the applicability of FRα-targeted agents in these patients.

EGFR targeted nanobody-photosensitizer conjugates for photodynamic therapy in a pre-clinical model of head and neck cancer.

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer (PS) and has been used to treat head and neck cancers. Yet, common PS lack tumor specificity, which leads to collateral damage to normal tissues. Targeted delivery of PS via antibodies has pre-clinically improved tumor selectivity. However, antibodies have long half-lives and relatively poor tissue penetration, which could limit therapeutic efficacy and lead to long photosensitivity. Here, in this feasibility study, we evaluate at the pre-clinical level a recently introduced format of targeted PDT, which employs nanobodies as targeting agents and a water-soluble PS (IRDye700DX) that is traceable through optical imaging. In vitro, the PS solely binds to cells and induces phototoxicity on cells overexpressing the epidermal growth factor receptor (EGFR), when conjugated to the EGFR targeted nanobodies. To investigate whether this new format of targeted PDT is capable of inducing selective tumor cell death in vivo, PDT was applied on an orthotopic mouse tumor model with illumination at 1h post-injection of the nanobody-PS conjugates, as selected from quantitative fluorescence spectroscopy measurements. In parallel, and as a reference, PDT was applied with an antibody-PS conjugate, with illumination performed 24h post-injection. Importantly, EGFR targeted nanobody-PS conjugates led to extensive tumor necrosis (approx. 90%) and almost no toxicity in healthy tissues, as observed through histology 24h after PDT. Overall, results show that these EGFR targeted nanobody-PS conjugates are selective and able to induce tumor cell death in vivo. Additional studies are now needed to assess the full potential of this approach to improving PDT.

Stromal Targets for Fluorescent-Guided Oncologic Surgery.

Pre-operative imaging techniques are essential for tumor detection and diagnosis, but offer limited help during surgery. Recently, the applicability of imaging during oncologic surgery has been recognized, using near-infrared fluorescent dyes conjugated to targeting antibodies, peptides, or other vehicles. Image-guided oncologic surgery (IGOS) assists the surgeFon to distinguish tumor from normal tissue during operation, and can aid in recognizing vital structures. IGOS relies on an optimized combination of a dedicated fluorescent camera system and specific probes for targeting. IGOS probes for clinical use are not widely available yet, but numerous pre-clinical studies have been published and clinical trials are being established or prepared. Most of the investigated probes are based on antibodies or peptides against proteins on the membranes of malignant cells, whereas others are directed against stromal cells. Targeting stroma cells for IGOS has several advantages. Besides the high stromal content in more aggressive tumor types, the stroma is often primarily located at the periphery/invasive front of the tumor, which makes stromal targets particularly suited for imaging purposes. Moreover, because stroma up-regulation is a physiological reaction, most proteins to be targeted on these cells are "universal" and not derived from a specific genetic variation, as is the case with many upregulated proteins on malignant cancer cells.

Inhibition of MicroRNA-494 Reduces Carotid Artery Atherosclerotic Lesion Development and Increases Plaque Stability.

OBJECTIVES: Unstable atherosclerotic lesions in carotid arteries require surgical endarterectomy to reduce the risk of ischemic stroke. We aimed to identify microRNAs that exert a broad effect on atherosclerotic plaque formation and stability in the carotid artery. BACKGROUND: We made a selection of 164 genes involved in atherosclerosis. Using www.targetscan.org, we determined which microRNAs potentially regulate expression of these genes. We identified multiple microRNAs from the 14q32 microRNA cluster, which is highly involved in vascular remodeling. In human plaques, collected during carotid endarterectomy surgery, we found that 14q32 microRNA (miR-494) was abundantly expressed in unstable lesions. METHODS: We induced atherosclerotic plaque formation in hypercholesterolemic ApoE mice by placing semiconstrictive collars around both carotid arteries. We injected "Gene Silencing Oligonucleotides" against miR-494 (GSO-494) or negative control (GSO-control). Using fluorescently labeled GSOs, we confirmed uptake of GSOs in affected areas of the carotids, but not elsewhere in the vasculature. RESULTS: After injection of GSO-494, we observed significant downregulation of miR-494 expression in the carotid arteries, although miR-494 target genes were upregulated. Further analyses revealed a 65% decrease in plaque size after GSO-494 treatment. Plaque stability was increased in GSO-494-treated mice, determined by an 80% decrease in necrotic core size and a 50% increase in plaque collagen content. Inhibition of miR-494 also resulted in decreased cholesterol levels and decreased very low-density lipoprotein (VLDL) fractions. CONCLUSIONS: Treatment with GSO-494 results in smaller atherosclerotic lesions with increased plaque stability. Inhibition of miR-494 may decrease the risk of surgical complications or even avert endarterectomy surgery in some cases.

uPAR-targeted multimodal tracer for pre- and intraoperative imaging in cancer surgery.

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.

Preclinical evaluation of a novel CEA-targeting near-infrared fluorescent tracer delineating colorectal and pancreatic tumors.

Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in nonradical resections, increased cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastrointestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA-targeted near-infrared fluorescent (NIRF) tracer, based on a disulfide-stabilized single-chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell-based plate assays and orthotopic colorectal (HT-29, well differentiated) and pancreatic (BXPC-3, poorly differentiated) xenogeneic human-mouse models. NIRF signals were visualized using the clinically compatible FLARE™ imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor-to-background ratios of 5.1 ± 0.6 at 72 hr postinjection, which proved suitable for intraoperative detection and delineation of tumor boarders and small (residual) tumor nodules in mice, between 8 and 96 hr postinjection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent-guided surgery applications. If successfully translated clinically, this tracer could help improve the completeness of surgery and thus survival.

Intraoperative near-infrared fluorescence imaging of a paraganglioma using methylene blue: A case report.

INTRODUCTION: Intraoperative identification of tumors can be challenging. Near-infrared (NIR) fluorescence imaging is an innovative technique that can assist in intraoperative identification of tumors, which may otherwise be undetectable. PRESENTATION OF CASE: A 19-year-old patient with symptoms, normetanephrine levels and radiological findings suspicious for a paraganglioma, a rare tumor arising from extra-adrenal chromaffin cells within the sympathetic nervous system, is presented. Intraoperative NIR fluorescence imaging using intravenous administration of methylene blue (MB) assisted in intraoperative detection of the tumor, and even identified a smaller second lesion, which was not identified during surgery by visual inspection. DISCUSSION: Although the exact mechanism of MB accumulation in neuroendocrine tumors is unclear, it is described in both preclinical and clinical studies. CONCLUSION: In this report, we describe the first case of intraoperative NIR fluorescence imaging of a paraganglioma using MB, which identified an otherwise undetectable lesion.

Current and future intraoperative imaging strategies to increase radical resection rates in pancreatic cancer surgery.

Prognosis of patients with pancreatic cancer is poor. Even the small minority that undergoes resection with curative intent has low 5-year survival rates. This may partly be explained by the high number of irradical resections, which results in local recurrence and impaired overall survival. Currently, ultrasonography is used during surgery for resectability assessment and frozen-section analysis is used for assessment of resection margins in order to decrease the number of irradical resections. The introduction of minimal invasive techniques in pancreatic surgery has deprived surgeons from direct tactile information. To improve intraoperative assessment of pancreatic tumor extension, enhanced or novel intraoperative imaging technologies accurately visualizing and delineating cancer cells are necessary. Emerging modalities are intraoperative near-infrared fluorescence imaging and freehand nuclear imaging using tumor-specific targeted contrast agents. In this review, we performed a meta-analysis of the literature on laparoscopic ultrasonography and we summarized and discussed current and future intraoperative imaging modalities and their potential for improved tumor demarcation during pancreatic surgery.

Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 increases neovascularization and blood flow recovery after ischemia.

RATIONALE: Effective neovascularization is crucial for recovery after cardiovascular events. OBJECTIVE: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation. METHODS AND RESULTS: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation. CONCLUSIONS: The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization.