Amino BODIPY-Based Blue Fluorescent Probes for Aldehyde Dehydrogenase 1-Expressing Cells.

Aldehyde dehydrogenase 1 (ALDH1) plays an important role as a stem cell marker. In the field of stem cell biology, a green fluorescent ALDH1 probe has been principally used, but there is a need for more options in probe color. We designed and synthesized two blue fluorescent ALDH1 probes using 8-amino BODIPY and aminomethylbenzaldehyde. These probes can be simultaneously used with other color probes. Here, we demonstrate successful examples of the simultaneous use of these probes with green fluorescent protein.

Development of Highly Selective Fluorescent Probe Enabling Flow-Cytometric Isolation of ALDH3A1-Positive Viable Cells.

Aldehyde dehydrogenase (ALDH) is overexpressed in some subpopulations of stem cells and cancer cells. We have designed and synthesized the first selective fluorescent probe for class 3 ALDH (ALDH3A1). This probe enabled the visualization of ALDH3A1-positive cells by fluorescence microscopy as well as flow-cytometric isolation of ALDH3A1-positive viable cells from a human Caucasian esophageal squamous cell line (OE21) that heterogeneously expresses ALDH3A1.

Epidermal growth factor receptor (EGFR) signaling regulates global metabolic pathways in EGFR-mutated lung adenocarcinoma.

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFRTKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.

Endoscopic findings using narrow-band imaging to distinguish between basal cell hyperplasia and carcinoma of the pharynx.

Narrow-band imaging (NBI) has been reported to be useful for detecting superficial-type esophageal or head and neck squamous cell carcinoma (SCC), and in the present study we have used NBI to detect non-carcinomatous lesions, such as basal cell hyperplasia (BCH) accompanied by microvascular irregularities; these non-carcinomatous lesions were pathologically discriminated from squamous cell carcinoma of the pharynx. The aim of the present study was to clarify the endoscopic characteristics of BCH that contribute to the discrimination of superficial-type head and neck SCC (HNSCC). We examined the key endoscopic findings capable of distinguishing BCH from SCC using 26 BCH and 37 superficial-type SCC of the pharynx that had been pathologically diagnosed at our institution between January 2008 and July 2012. The clinicopathological factors were also compared. The size of the BCH lesions was significantly smaller (P < 0.001), and their intervascular transparency was more clearly observed (P < 0.001). Intra-epithelial papillary capillary loop (IPCL) shapes were less variable and monotonous (P < 0.001), and the distribution of the IPCL was more regular with an interval comparable to that of SCC (P < 0.001), although no significant differences in the sharpness of the lesion border, dilatation of IPCL and tortuosity of the IPCL were seen between the BCH and SCC lesions. This study revealed that BCH was an independent entity in terms of not only pathological findings, but also endoscopic findings observed using NBI, such as the regular distribution of IPCL and the preserved intervascular transparency.

CdTe XG-Cam: A new high-resolution x-ray and gamma-ray camera for studies of the pharmacokinetics of radiopharmaceuticals in small animals.

BACKGROUND: The recent emergence of targeted radionuclide therapy has increased the demand for imagers capable of visualizing pharmacokinetics in developing radiopharmaceuticals in the preclinical phase. Some radionuclides emit hard x-rays and gamma-rays below 100 keV, in which energy range the performance of conventional NaI scintillators is poor. Multipinhole collimators are also used for small animal imaging with a good spatial resolution but have a limited field of view (FOV). PURPOSE: In this study, a new imager with high sensitivity over a wide FOV in the low-energy band ( < $<$ 100 keV) was developed for the pharmacokinetic study. METHODS: We developed an x-ray and gamma-ray camera for high-resolution spectroscopy, named "CdTe XG-Cam," equipped with a cadmium telluride semiconductor detector and a parallel-hole collimator using a metal 3D printer. To evaluate the camera-system performance, phantom measurements with single and dual nuclides ( 99 m Tc $^{\rm 99m}{\rm Tc}$ , 111 In $^{111}{\rm In}$ , and 125 I ) $^{125}{\rm I)}$ were performed. The performance for in vivo imaging was evaluated using tumor-bearing mice to which a nuclide ( 99 m Tc $^{\rm 99m}{\rm Tc}$ or 125 I ) $^{125}{\rm I)}$  administered. RESULTS: We simultaneously obtained information on 111 In $^{111}{\rm In}$ and 125 I $^{125}{\rm I}$ , which emit emission lines in the low-energy band with peak energies close to each other (23-26 keV for 111 In $^{111}{\rm In}$ and 27-31 keV for 125 I ) $^{125}{\rm I)}$ , and applied an analytical method based on spectral model fitting to determine the individual radioactivities accurately. In the small animal imaging, the distributions of the nuclide in tumors were accurately quantified and time-activity curves in tumors are obtained. CONCLUSIONS: The demonstrated capability of our system to perform in vivo imaging suggests that the camera can be used for applications of pharmacokinetics research.

Dual-radionuclide in vivo imaging of micro-metastasis and lymph tract with submillimetre resolution.

Multi-radionuclide in vivo imaging with submillimetre resolution can be a potent tool for biomedical research. While high-resolution radionuclide imaging faces challenges in sensitivity, multi-radionuclide imaging encounters difficulty due to radiation contamination, stemming from crosstalk between radionuclides and Compton scattering. Addressing these challenges simultaneously is imperative for multi-radionuclide high-resolution imaging. To tackle this, we developed a high-spatial-resolution and high-energy-resolution small animal single-photon emission computed tomography (SPECT) scanner, named CdTe-DSD SPECT-I. We first assessed the feasibility of multi-tracer SPECT imaging of submillimetre targets. Using the CdTe-DSD SPECT-I, we performed SPECT imaging of submillimetre zeolite spheres absorbed with 125I- and subsequently imaged 125I-accumulated spheroids of 200-400 µm in size within an hour, achieving clear and quantitative images. Furthermore, dual-radionuclide phantom imaging revealed a distinct image of the submillimetre sphere absorbed with 125I- immersed in a 99mTc-pertechnetate solution, and provided a fair quantification of each radionuclide. Lastly, in vivo imaging was conducted on a cancer-bearing mouse with lymph node micro-metastasis using dual-tracers. The results displayed dual-tracer images of lymph tract by 99mTc-phytic acid and the submillimetre metastatic lesion by 125I-, shown to align with the immunofluorescence image.

Development and Utility of an Imaging System for Internal Dosimetry of Astatine-211 in Mice.

In targeted radionuclide therapy, determining the absorbed dose of the ligand distributed to the whole body is vital due to its direct influence on therapeutic and adverse effects. However, many targeted alpha therapy drugs present challenges for in vivo quantitative imaging. To address this issue, we developed a planar imaging system equipped with a cadmium telluride semiconductor detector that offers high energy resolution. This system also comprised a 3D-printed tungsten collimator optimized for high sensitivity to astatine-211, an alpha-emitting radionuclide, and adequate spatial resolution for mouse imaging. The imager revealed a spectrum with a distinct peak for X-rays from astatine-211 owing to the high energy resolution, clearly distinguishing these X-rays from the fluorescent X-rays of tungsten. High collimator efficiency (4.5 × 10-4) was achieved, with the maintenance of the spatial resolution required for discerning mouse tissues. Using this system, the activity of astatine-211 in thyroid cancer tumors with and without the expression of the sodium iodide symporter (K1-NIS/K1, respectively) was evaluated through in vivo imaging. The K1-NIS tumors had significantly higher astatine-211 activity (sign test, p = 0.031, n = 6) and significantly decreased post-treatment tumor volume (Student's t-test, p = 0.005, n = 6). The concurrent examination of intratumor drug distribution and treatment outcome could be performed with the same mice.

Simultaneous visualization of multiple radionuclides in vivo.

The insufficient energy and spatial resolutions of radionuclide imaging with conventional scintillation detectors restrict the visualization of multiple radionuclides and of microstructures in tissue. Here we report the development and performance of an imaging system equipped with a cadmium telluride diode detector that achieves an energy resolution of 1.7% at 140 keV and a spatial resolution of 250 µm. The combination of high-resolution spectra fitted to an X-ray analysis model of the emission lines of the radionuclides in a chosen energy band allowed us to accurately determine individual radiation activities from three radionuclides to simultaneously visualize thyroid tissue (via intravenously administered iodine-125), mandibular lymph nodes (via the intramuscular injection of indium-111) and parotid lymph nodes (via a subcutaneous injection of technetium-99m) in mice. Multi-radionuclide imaging may find advantageous applications in biomedical imaging.

Difference in the relative biological effectiveness and DNA damage repair processes in response to proton beam therapy according to the positions of the spread out Bragg peak.

BACKGROUND: Cellular responses to proton beam irradiation are not yet clearly understood, especially differences in the relative biological effectiveness (RBE) of high-energy proton beams depending on the position on the Spread-Out Bragg Peak (SOBP). Towards this end, we investigated the differences in the biological effect of a high-energy proton beam on the target cells placed at different positions on the SOBP, using two human esophageal cancer cell lines with differing radiosensitivities. METHODS: Two human esophageal cancer cell lines (OE21, KYSE450) with different radiosensitivities were irradiated with a 235-MeV proton beam at 4 different positions on the SOBP (position #1: At entry; position #2: At the proximal end of the SOBP; position #3: Center of the SOBP; position #4: At the distal end of the SOBP), and the cell survivals were assessed by the clonogenic assay. The RBE10 for each position of the target cell lines on the SOBP was determined based on the results of the cell survival assay conducted after photon beam irradiation. In addition, the number of DNA double-strand breaks was estimated by quantitating the number of phospho-histone H2AX (γH2AX) foci formed in the nuclei by immunofluorescence analysis. RESULTS: In regard to differences in the RBE of a proton beam according to the position on the SOBP, the RBE value tended to increase as the position on the SOBP moved distally. Comparison of the residual number of γH2AX foci at the end 24 h after the irradiation revealed, for both cell lines, a higher number of foci in the cells irradiated at the distal end of the SOPB than in those irradiated at the proximal end or center of the SOBP. CONCLUSIONS: The results of this study demonstrate that the RBE of a high-energy proton beam and the cellular responses, including the DNA damage repair processes, to high-energy proton beam irradiation, differ according to the position on the SOBP, irrespective of the radiosensitivity levels of the cell lines.

Risk factors for intraoperative perforation during endoscopic submucosal dissection of superficial esophageal squamous cell carcinoma.

AIM: To identify the risk factors and clarify the subsequent clinical courses. METHODS: This study retrospectively analyzed consecutive patients with esophageal squamous cell carcinoma (ESCC) treated using endoscopic submucosal dissection (ESD) between April 2008 and October 2012. We divided the ESCC lesions into perforation cases and non-perforation cases, and compared characteristics and endoscopic findings between the two groups. "Intraoperative perforation" was defined as the detection of a perforation site during ESD and the presence of mediastinal emphysema. RESULTS: In total, 147 patients with 156 ESCC lesions were treated by ESD. Intraoperative perforation was recorded for nine lesions (5.8%) from nine patients. Multivariate analysis identified mucosal deficiency larger than 75% of the circumference of the esophagus as an independent risk factor for intraoperative perforation (OR = 7.37, 95%CI: 1.45-37.4, P = 0.016). The predominant site of perforation was the left wall [6/9 (67%)]. Six of nine perforation sites were successfully closed by clips during the procedures. Two of nine cases required drainage for pleural effusions; however, all nine cases recovered with conservative treatment and without surgical intervention. At the median follow up of 42 mo after ESD, no cases of local recurrence or distant organ metastasis had been observed. CONCLUSION: This study suggests that mucosal deficiency larger than 75% of the luminal circumference is a risk factor for intraoperative perforation during ESD for ESCC.

Effect of novel bright image enhanced endoscopy using blue laser imaging (BLI).

BACKGROUND AND STUDY AIMS: The novel method of image-enhanced endoscopy (IEE) named blue laser imaging (BLI) can enhance the contrast of surface vessels using lasers for light illumination. BLI has two IEE modes: high contrast mode (BLI-contrast) for use with magnification, and bright mode (BLI-bright), which achieves a brighter image than BLI-contrast and yet maintains the enhanced visualization of vascular contrast that is expected for the detection of tumors from a far field of view. The aim of this study is to clarify the effect of BLI-bright with a far field of view compared to BLI-contrast and commonly available narrow-band imaging (NBI). PATIENTS AND METHODS: Patients with neoplasia, including early cancer in the pharynx, esophagus, stomach, or colorectum, were recruited and underwent tandem endoscopy with BLI and NBI systems. Six sets of images of the lesions were captured with a changing observable distance from 3 to 40 mm. Individual sets of images taken from various observable distances were assessed for visibility among BLI-bright, BLI-contrast, and NBI modes. The brightness and contrast of these images were also analyzed quantitatively. RESULTS: Of 51 patients, 39 were assessed. Image analysis indicated that only BLI-bright maintained adequate brightness and contrast up to 40 mm and had significantly longer observable distances compared to the other methods. Furthermore, BLI-bright enhanced the visualization of serious lesions infiltrating into deeper layers, such as esophageal lamina propria or gastric submucosal cancers. CONCLUSIONS: BLI-bright will be a helpful tool for the far-field view with IEE in organs with wider internal spaces such as the stomach.

Percutaneous endoscopic gastrostomy using the direct method for aerodigestive cancer patients.

BACKGROUND: In esophageal or head and neck cancer patients, percutaneous endoscopic gastrostomy (PEG) by the pull method has higher complication rates than in noncancer patients. An introducer method is considered suitable for these patients, however, complications associated with deflation or rupture of the balloon-anchoring system are reported. AIM: To investigate the efficacy of a modified introducer method, called direct method, for aerodigestive cancer patients. MATERIALS AND METHODS: Between October 2007 and December 2010, direct-method PEG was performed in 160 patients with advanced-stage esophageal (59 patients) or head and neck (101 patients) cancer. Complications and postoperative course were retrospectively retrieved from charts. RESULTS: An ultrathin endoscopy was used in 109 patients (72.7%), and 23 patients received previous endoscopic dilatation (14%) due to the stricture caused by the tumor. The success rate of tube placement was 98.8% (158/160). Major complications related to tube insertion, such as panperitonitis, gastrointestinal hemorrhage, and postoperative pneumonia occurred in five patients (3.2%). One patient died for panperitonitis due to late-onset tube dislodgement. There were no cases of tube blockade and no metastases in the PEG site during the median follow-up period of 153 days. CONCLUSION: This was the first report of direct-method PEG in a number of esophageal or head and neck cancer patients. This method is optimal for its high success rate, acceptable safety profile, and little risk of stomal metastasis.

Gastrointestinal stromal tumors of the duodenum: a report of four cases.

Here, we report four cases of duodenal gastrointestinal stromal tumors (GISTs). In three patients, endoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS FNAB) was performed, and in one patient, the tumor tissue was sampled via forceps biopsy. In all the cases, GISTs could be diagnosed by histopathological examination of the biopsy samples. Ki-67 labeling indexes (Ki-67 LIs) of the 3 EUS FNAB samples were very low. It should be noted that a sufficient amount of tissue sample could not be obtained in one of these cases. In two patients who underwent laparotomy, the Ki-67 LIs of the biopsy samples corresponded with the Ki-67 LIs of the surgically resected samples and were also consistent with the tumor risk grade. In two patients who did not undergo laparotomy, the low Ki-67 LIs corresponded with a good clinical course.