Intraoperative fluorescence delineation of head and neck cancer with a fluorescent anti-epidermal growth factor receptor nanobody.

Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti-EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12-800CW and R2-800CW). Orthotopic tongue tumors were induced in nude mice using the OSC-19-luc2-cGFP cell line. Tumor-bearing mice were injected with 25 µg 7D12-800CW, R2-800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12-800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12-800CW. A significantly higher tumor-to-background ratio (TBR) was observed in mice injected with 7D12-800CW compared to mice injected with R2-800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR-specific nanobody. After injection of 75 µg 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR-targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.

Optimization of sentinel lymph node mapping in bladder cancer using near-infrared fluorescence imaging.

BACKGROUND AND OBJECTIVES: Unlike other cancers, the Sentinel Lymph Node (SLN) procedure in bladder cancer requires special attention to the injection technique. The aim of this study was to assess feasibility and to optimize tracer injection technique for SLN mapping in bladder cancer patients using NIR fluorescence imaging. METHODS: Twenty patients with invasive bladder cancer scheduled for radical cystectomy were prospectively enrolled. Indocyanine green (ICG) bound to human serum albumin (complex ICG:HSA; 500 µM) was injected peritumourally to permit SLN mapping. ICG:HSA was first administrated serosally (n = 5), and subsequently mucosally by cystoscopic injection (n = 15). In the last cohort of 12 patients treated with cystoscopic injection, the bladder was kept filled with saline for at least 15 min. RESULTS: Fluorescent lymph nodes were observed only in the patient group with cystoscopic injection of ICG:HSA. Filling of the bladder post-injection was of added value to promote drainage of ICG:HSA to the lymph nodes, and in 11 of these 12 patients (92%) one or more NIR fluorescent lymph nodes were identified. CONCLUSIONS: The current study demonstrates proof-of-principle of using NIR fluorescence imaging for SLN identification in bladder cancer. Cystoscopic injection with distension of the bladder appears optimal for SLN mapping.

Clinical trial of combined radio- and fluorescence-guided sentinel lymph node biopsy in breast cancer.

BACKGROUND: Combining radioactive colloids and a near-infrared (NIR) fluorophore permits preoperative planning and intraoperative localization of deeply located sentinel lymph nodes (SLNs) with direct optical guidance by a single lymphatic tracer. The aim of this clinical trial was to evaluate and optimize a hybrid NIR fluorescence and radioactive tracer for SLN detection in patients with breast cancer. METHODS: Patients with breast cancer undergoing SLN biopsy were enrolled. The day before surgery, a periareolar injection of indocyanine green (ICG)-99mTc-radiolabelled nanocolloid was administered and a lymphoscintigram acquired. Blue dye was injected immediately before surgery. Intraoperative SLN localization was performed using a γ probe and the Mini-FLARE™ NIR fluorescence imaging system. Patients were divided into two dose groups, with one group receiving twice the particle density of ICG and nanocolloid, but the same dose of radioactive 99mTc. RESULTS: Thirty-two patients were enrolled in the trial. At least one SLN was identified before and during operation. All 48 axillary SLNs could be detected by γ tracing and NIR fluorescence imaging, but only 42 of them stained blue. NIR fluorescence imaging permitted detection of lymphatic vessels draining to the SLN up to 29 h after injection. Doubling the particle density did not yield a difference in fluorescence intensity (median 255 (range 98-542) versus 284 (90-921) arbitrary units; P = 0.590) or signal-to-background ratio (median 5·4 (range 3·0-15·4) versus 4·9 (3·5-16·3); P = 1·000) of the SLN. CONCLUSION: The hybrid NIR fluorescence and radioactive tracer permitted accurate preoperative and intraoperative detection of the SLNs in patients with breast cancer. REGISTRATION NUMBER: NTR3685 (Netherlands Trial Register; http://www.trialregister.nl).

Dose optimization for near-infrared fluorescence sentinel lymph node mapping in patients with melanoma.

BACKGROUND: Regional lymph node involvement is the most important prognostic factor in cutaneous melanoma. As only 20% of patients with melanoma have occult nodal disease and would benefit from a regional lymphadenectomy, the sentinel lymph node (SLN) biopsy was introduced. Near-infrared (NIR) fluorescence has been hypothesized to improve SLN mapping. OBJECTIVES: To assess the potential of intraoperative NIR fluorescence imaging to improve SLN mapping in patients with melanoma and to examine the optimal dose of indocyanine green adsorbed to human serum albumin (ICG:HSA). METHODS: Fifteen consecutive patients with cutaneous melanoma underwent the standard SLN procedure using (99m) technetium-nancolloid and patent blue. In addition, intraoperative NIR fluorescence imaging was performed after injection of 1·6 mL of 600, 800, 1000 or 1200 µmolL(-1) of ICG: HSA in four quadrants around the primary excision scar. RESULTS: NIR fluorescence SLN mapping was successful in 93% of patients. In one patient, no SLN could be identified using either conventional methods or NIR fluorescence. A total of 30 SLNs (average 2·0, range 1-7) were detected, 30 radioactive (100%), 27 blue (73%) and 30 NIR fluorescent (100%). With regard to the effect of concentration on signal-to-background ratios a trend (P=0·066) was found favouring the 600, 800 and 1000 µmol L(-1) groups over the 1200 µmol L(-1) group. CONCLUSION: This study demonstrates feasibility and accuracy of SLN mapping using ICG: HSA. Considering safety, cost and pharmacological characteristics, an ICG: HSA concentration of 600 µmolL(-1) appears optimal for SLN mapping in cutaneous melanoma, although lower doses need to be assessed.

Near-infrared fluorescence sentinel lymph node biopsy in vulvar cancer: a randomised comparison of lymphatic tracers.

This study aims to confirm the feasibility of near-infrared (NIR) fluorescence imaging for sentinel lymph node (SLN) biopsy in vulvar cancer and to compare the tracer indocyanine green (ICG) bound to human serum albumin (HSA) versus ICG alone. Women received 99mTc-nanocolloid and patent blue for SLN detection. Subsequently, women randomly received ICG:HSA or ICG alone. In 24 women, 35 SLNs were intraoperatively detected. All SLNs detected were radioactive and NIR fluorescent and 27 (77%) were blue. No significant difference was found between ICG:HSA and ICG alone. This trial confirms the feasibility of NIR fluorescence imaging for SLN mapping in vulvar cancer.

Predicting the survival of experimental ischaemic small bowel using intraoperative near-infrared fluorescence angiography.

BACKGROUND: Predicting the long-term viability of ischaemic bowel during surgery is challenging. The aim was to determine whether intraoperative near-infrared angiography (NIR-AG) of ischaemic bowel might provide metrics that were predictive of long-term outcome. METHODS: NIR-AG using indocyanine green was performed on 24 pigs before, and after inducing bowel ischaemia to determine the feasibility of NIR-AG for detecting compromised perfusion. Contrast-to-background ratio (CBR) over time was measured in regions of interest throughout the bowel, and various metrics of the CBR-time curve were developed. Sixty rat small bowels, with or without strangulation, were imaged during surgery and on day 3 after operation. CBR metrics and clinical findings were assessed quantitatively for their ability to predict animal survival, histological grade of ischaemic injury and visible necrosis on day 3. RESULTS: In ischaemic pig small bowel, various qualitative and quantitative CBR metrics appeared to correlate with bowel injury as a function of distance from normal bowel. In rats, intraoperative clinical assessment showed high specificity but low sensitivity for predicting outcome on day 3 after operation. Qualitative patterns of the CBR-time curve, such as absence of an arterial inflow peak and presence of a NIR filling defect, resulted in better prediction of survival (90 per cent), histological grade (85 per cent) and visible necrosis on day 3 (92 per cent). CONCLUSION: Survival of ischaemic bowel was predicted by intraoperative NIR-AG with greater accuracy than clinical evaluation alone.

Near-infrared fluorescence imaging in patients undergoing pancreaticoduodenectomy.

BACKGROUND: Intraoperative visualization of pancreatic tumors has the potential to improve radical resection rates. Intraoperative visualization of the common bile duct and bile duct anastomoses could be of added value. In this study, we explored the use of indocyanine green (ICG) for these applications and attempted to optimize injection timing and dose. METHODS: Eight patients undergoing a pancreaticoduodenectomy were injected intravenously with 5 or 10 mg ICG. During and after injection, the pancreas, tumor, common bile duct and surrounding organs were imaged in real time using the Mini-FLARE™ near-infrared (NIR) imaging system. RESULTS: No clear tumor-to-pancreas contrast was observed, except for incidental contrast in 1 patient. The common bile duct was clearly visualized using NIR fluorescence, within 10 min after injection, with a maximal contrast between 30 and 90 min after injection. Patency of biliary anastomoses could be visualized due to biliary excretion of ICG. CONCLUSION: No useful tumor demarcation could be visualized in pancreatic cancer patients after intravenous injection of ICG. However, the common bile duct and biliary anastomoses were clearly visualized during the observation period. Therefore, these imaging strategies could be beneficial during biliary surgery in cases where the surgical anatomy is aberrant or difficult to identify.

Control of intracellular pH and growth by fibronectin in capillary endothelial cells.

The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.

Minimal activators that bind to the KIX domain of p300/CBP identified by phage display screening.

Human gene therapy approaches involving transcription factors often rely on artificial activation domains for transcriptional activation. These domains are often large (e.g., 80 amino acids for VP16), recruit multiple co-activation complexes at once, and offer no fine control over the level of transcription. In an attempt to understand the sequence and structural requirements of a minimal mammalian activator, we employed a molecular diversity approach with a peptide phage display library composed of random eight-amino acid peptides. Using the KIX domain of the mammalian co-activators p300 and CBP as target, we discovered a family of synthetic binding peptides. These peptides share significant homology with natural KIX domain ligands, and are shown to bind an overlapping, yet distinct, surface of p300/CREB-binding protein (CBP). When fused to a heterologous DNA binding domain, these synthetic peptides function as titratable, modular, and potent transcriptional activators in living cells through specific recruitment of p300/CBP, with the level of transcriptional activation proportional to the affinity of the synthetic peptide for the KIX domain. Taken together, our data demonstrate that a molecular diversity approach can be used to discover minimal, co-activator domain-specific synthetic activators, and that transcriptional activation can be modulated as desired at the level of co-activator recruitment.

In vivo near-infrared fluorescence imaging of osteoblastic activity.

In vertebrates, the development and integrity of the skeleton requires hydroxyapatite (HA) deposition by osteoblasts. HA deposition is also a marker of, or a participant in, processes as diverse as cancer and atherosclerosis. At present, sites of osteoblastic activity can only be imaged in vivo using gamma-emitting radioisotopes. The scan times required are long, and the resultant radioscintigraphic images suffer from relatively low resolution. We have synthesized a near-infrared (NIR) fluorescent bisphosphonate derivative that exhibits rapid and specific binding to HA in vitro and in vivo. We demonstrate NIR light-based detection of osteoblastic activity in the living animal, and discuss how this technology can be used to study skeletal development, osteoblastic metastasis, coronary atherosclerosis, and other human diseases.

Long-term follow-up after near-infrared fluorescence-guided resection of colorectal liver metastases: A retrospective multicenter analysis.

BACKGROUND: Several studies demonstrated that intraoperative near-infrared fluorescence (NIRF) imaging using indocyanine green (ICG) identifies (sub)capsular colorectal liver metastases (CRLM) missed by other techniques. It is unclear if this results in any survival benefit. This study evaluates long-term follow-up after NIRF-guided resection of CRLM using ICG. METHODS: First, patients undergoing resection of CRLM with or without NIRF imaging were analyzed retrospectively. Perioperative details, liver-specific recurrence-free interval and overall survival were compared. Second, the prognosis of patients in whom additional metastases were identified solely by NIRF was studied. RESULTS: Eighty-six patients underwent resection with NIRF imaging and 87 without. In significantly more patients of the NIRF imaging cohort additional metastases were identified during surgery (25% vs. 13%, p = 0.04). Tumors identified solely by NIRF imaging were significantly smaller compared to additional metastases identified also by inspection, palpation or intraoperative ultrasound (3.2 ± 1.8 mm vs. 7.4 ± 2.6 mm, p < 0.001). Liver-specific recurrence-free survival at 4 years was 47% with NIRF imaging and 39% without (hazard ratio at multivariate analysis 0.73, 95% CI 0.42-1.28, p = 0.28). Overall survival at 4 years was 62% and 59%, respectively (p = 0.79). No liver recurrences occurred within 3 years follow-up in 52% of patients in whom additional metastases were resected based on only NIRF imaging. CONCLUSIONS: This study suggests that NIRF imaging identifies significantly more and smaller tumors during resection of CRLM, preventing recurrences in a subset of patients. Given its safety profile and low expense, routine use can be considered until tumor targeting fluorescent tracers are clinically available.

Cytoplasmic pH and anchorage-independent growth induced by v-Ki-ras, v-src or polyoma middle T.

We had previously shown that spreading of normal cells on tissue culture plastic coated with extracellular matrix (ECM) proteins led to an increase in cytoplasmic pH (pHi). Since alkalinization of the cytoplasm is associated with activation of a number of signaling pathways that control growth, and is itself required for cell growth, we proposed that this phenomenon could explain, at least in part, why growth of normal cells is anchorage-dependent. Preliminary results showed that pHi in cells transformed by the ras or src oncogenes had an alkaline pHi even when completely round. To further explore the relationship between pHi and anchorage-independent growth, a series of cells transformed by mutants of the polyoma middle T oncogene, and a series of ras-transformed cells and revertants were examined. Growth in methyl cellulose was assayed, and pHi in both maximally spread and completely round cells was measured for each cell line. We found that all of the normal cells required spreading to maintain an alkaline pHi, whereas transformed cell lines had an alkaline pHi independent of spreading. There was a strong correlation between pHi in round cells and anchorage-independent growth. Thus, some plasma membrane oncogenes can substitute for cell spreading on EMC to raise pHi as well as to promote growth. These results may be relevant to understanding why transformation leads to changes not only in cellular requirements for growth factors, but also for anchorage.

High frequency of retinoic acid receptor beta abnormalities in human lung cancer.

One of the three human retinoic acid receptors, RAR-beta, maps to a region on the short arm of chromosome 3 frequently deleted in lung cancer. Because retinoic acid is required for normal epithelial cell growth and regulation, and loss of a retinoic acid receptor might be expected to contribute to oncogenesis, we examined RAR-beta RNA and DNA in normal lung, 33 lung cancer cell lines and nine primary lung tumors. Normally, RAR-beta is expressed as two transcripts, of sizes 3.1 kb and 2.8 kb, which are strongly induced by retinoic acid. At least 50% of the cell lines and 30% of the tumor samples show altered RAR-beta expression and/or inducibility, including examples of absence or specific loss of one of the RAR-beta transcripts. Abnormalities in the expression patterns of RAR-alpha and RAR-gamma also are found, but at a lower frequency than RAR-beta abnormalities. Southern analysis reveals alteration of the RAR-beta gene in three of the cell lines. Our data suggest that abnormalities in structure and expression of the RAR-beta gene may be involved in the pathogenesis of lung cancer.

An operational near-infrared fluorescence imaging system prototype for large animal surgery.

Near-infrared (NIR) fluorescence imaging has the potential to revolutionize human cancer surgery by providing sensitive, specific, and real-time intraoperative visualization of normal and disease processes. We have previously introduced the concept of a low-cost, safe, and easy-to-use NIR fluorescence imaging system that permits the surgeon to "see" surgical anatomy and NIR fluorescence simultaneously, non-invasively, with high spatial resolution, in real-time, and without moving parts. In this study, we present an operational prototype designed specifically for use during large animal surgery. Such a system serves as a foundation for future clinical studies. We discuss technical considerations, and provide details of the implementation of subsystems related to excitation light, light collection, computer, and software. Using the prototype, and the clinically available NIR fluorophore indocyanine green, we demonstrate vascular imaging in 35 kg pigs. Cancer-specific applications of this imaging system include image-guided cancer resection with real-time assessment of surgical margins, image-guided sentinel lymph node mapping, intraoperative mapping of tumor and normal vasculature, image-guided avoidance of critical structures such as nerves, and intraoperative detection of occult metastases in the surgical field. Taken together, this study describes an optical imaging system engineered for eventual translation to the clinic.

Real-time intraoperative detection of breast cancer using near-infrared fluorescence imaging and Methylene Blue.

BACKGROUND: Despite recent developments in preoperative breast cancer imaging, intraoperative localization of tumor tissue can be challenging, resulting in tumor-positive resection margins during breast conserving surgery. Based on certain physicochemical similarities between Technetium((99m)Tc)-sestamibi (MIBI), an SPECT radiodiagnostic with a sensitivity of 83-90% to detect breast cancer preoperatively, and the near-infrared (NIR) fluorophore Methylene Blue (MB), we hypothesized that MB might detect breast cancer intraoperatively using NIR fluorescence imaging. METHODS: Twenty-four patients with breast cancer, planned for surgical resection, were included. Patients were divided in 2 administration groups, which differed with respect to the timing of MB administration. N = 12 patients per group were administered 1.0 mg/kg MB intravenously either immediately or 3 h before surgery. The mini-FLARE imaging system was used to identify the NIR fluorescent signal during surgery and on post-resected specimens transferred to the pathology department. Results were confirmed by NIR fluorescence microscopy. RESULTS: 20/24 (83%) of breast tumors (carcinoma in N = 21 and ductal carcinoma in situ in N = 3) were identified in the resected specimen using NIR fluorescence imaging. Patients with non-detectable tumors were significantly older. No significant relation to receptor status or tumor grade was seen. Overall tumor-to-background ratio (TBR) was 2.4 ± 0.8. There was no significant difference between TBR and background signal between administration groups. In 2/4 patients with positive resection margins, breast cancer tissue identified in the wound bed during surgery would have changed surgical management. Histology confirmed the concordance of fluorescence signal and tumor tissue. CONCLUSIONS: This feasibility study demonstrated an overall breast cancer identification rate using MB of 83%, with real-time intraoperative guidance having the potential to alter patient management.

Intraoperative detection of cell injury and cell death with an 800 nm near-infrared fluorescent annexin V derivative.

The intraoperative detection of cell injury and cell death is fundamental to human surgeries such as organ transplantation and resection. Because of low autofluorescence background and relatively high tissue penetration, invisible light in the 800 nm region provides sensitive detection of disease pathology without changing the appearance of the surgical field. In order to provide surgeons with real-time intraoperative detection of cell injury and death after ischemia/reperfusion (I/R), we have developed a bioactive derivative of human annexin V (annexin800), which fluoresces at 800 nm. Total fluorescence yield, as a function of bioactivity, was optimized in vitro, and final performance was assessed in vivo. In liver, intestine and heart animal models of I/R, an optimal signal to background ratio was obtained 30 min after intravenous injection of annexin800, and histology confirmed concordance between planar reflectance images and actual deep tissue injury. In summary, annexin800 permits sensitive, real-time detection of cell injury and cell death after I/R in the intraoperative setting, and can be used during a variety of surgeries for rapid assessment of tissue and organ status.

Three catheter-based strategies for cardiac delivery of therapeutic gelatin microspheres.

Gelatin hydrogel microspheres (GHMs) have been reported as novel non-viral vectors for gene or protein delivery (GHM therapy). However, the components of an effective catheter-based delivery strategy for GHM therapy are unknown. We evaluated the effectiveness of three catheter-based strategies for cardiac GHM therapy: (1) antegrade injection (AI) via coronary arteries; (2) retrograde injection (RI) via coronary veins; and (3) direct myocardial injection (DI) via the coronary sinus. AI distributed microspheres homogeneously throughout the target area with 73+/-11% retention. RI scattered microspheres non-homogenously with 22+/-8% retention. DI distributed microspheres in the needle-advanced area with 47+/-14% retention. However, despite high efficiency, AI did not show biological effects of inducing angiogenesis from basic fibroblast growth factor bound to GHMs. Furthermore, focal micro-infarctions, owing to micro-embolism of aggregated GHMs into small coronary arterioles, were detected in the AI group. Conversely, only RI and DI groups displayed increased coronary flow reserve. DI groups also demonstrated increased capillary density. These results suggest that RI and DI are effective for cardiac GHM therapy, while AI appears inappropriate owing to the risk of focal infarctions.

Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins.

The pGEX glutathione S-transferase (GST) fusion protein system is used extensively for high level expression and rapid purification of fusion proteins from bacterial and eukaryotic cell lysates. Unfortunately, many GST fusion proteins are partially or completely insoluble, and thus cannot be purified efficiently from a crude lysate. We have adapted a protocol, previously used to solubilize actin, for the purification of otherwise insoluble GST fusion proteins. Using a GST fusion of the nontransmembrane protein tyrosine phosphatase 1B, we demonstrate that tyrosine phosphatase enzymatic activity is maintained during the purification process. We provide methods for the purification of GST fusion proteins at analytical and preparative scales, and demonstrate that saturation of glutathione agarose is dependent on fusion protein molecular weight. Finally, we present strategies for eluting purified fusion proteins from glutathione agarose beads, for storing eluted protein, and for preparing covalently coupled affinity matrices.

Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal.

We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.