Monitoring Pancreatic Carcinogenesis by the Molecular Imaging of Cathepsin E <i>In Vivo</i> Using Confocal Laser Endomicroscopy

<div><p>The monitoring of pancreatic ductal adenocarcinoma (PDAC) in high-risk populations is essential. Cathepsin E (CTSE) is specifically and highly expressed in PDAC and pancreatic intraepithelial neoplasias (PanINs), and its expression gradually increases along with disease progression. In this study, we first established an <i>in situ</i> 7,12-dimethyl-1,2-benzanthracene (DMBA)-induced rat model for PanINs and PDAC and then confirmed that tumorigenesis properties in this model were consistent with those of human PDAC in that CTSE expression gradually increased with tumor development using histology and immunohistochemistry. Then, using <i>in vivo</i> imaging of heterotopically implanted tumors generated from CTSE- overexpressing cells (PANC-1-CTSE) in nude mice and <i>in vitro</i> imaging of PanINs and PDAC in DMBA-induced rats, the specificity of the synthesized CTSE-activatable probe was verified. Quantitative determination identified that the fluorescence signal ratio of pancreatic tumor to normal pancreas gradually increased in association with progressive pathological grades, with the exception of no significant difference between PanIN-II and PanIN-III grades. Finally, we monitored pancreatic carcinogenesis <i>in vivo</i> using confocal laser endomicroscopy (CLE) in combination with the CTSE-activatable probe. A prospective double-blind control study was performed to evaluate the accuracy of this method in diagnosing PDAC and PanINs of all grades (>82.7%). This allowed us to establish effective diagnostic criteria for CLE in PDAC and PanINs to facilitate the monitoring of PDAC in high-risk populations.</p></div

Assessment of two researchers on the CLE images, including the average sensitivity, specificity, positive predictive value, negative predictive value and accuracy.

<p>Abbreviations: CLE, confocal laser endomicroscopy; PPV, positive predictive value; NPV, negative predictive value.</p><p>Assessment of two researchers on the CLE images, including the average sensitivity, specificity, positive predictive value, negative predictive value and accuracy.</p

Quantitative determination curve of the <i>in vitro</i> imaging fluorescence signal of DMBA-induced PDAC and PanINs in rats injected with probe A and probe a.

<p>The results showed that the fluorescent signal ratio of tumor:normal pancreatic parenchyma increased with progressive pathological grade in rats injected with probe A. On the other hand, the ratio was almost unchanged in rats injected with probe a. Error bars represent SD. N = normal.</p

Genome-Wide Assessment in Escherichia coli Reveals Time-Dependent Nanotoxicity Paradigms

The use of engineered nanomaterials (eNM) in consumer and industrial products is increasing exponentially. Our ability to rapidly assess their potential effects on human and environmental health is limited by our understanding of nanomediated toxicity. High-throughput screening (HTS) enables the investigation of nanomediated toxicity on a genome-wide level, thus uncovering their novel mechanisms and paradigms. Herein, we investigate the toxicity of zinc-containing nanomaterials (Zn-eNMs) using a time-resolved HTS methodology in an arrayed Escherichia coli genome-wide knockout (KO) library. The library was screened against nanoscale zerovalent zinc (nZn), nanoscale zinc oxide (nZnO), and zinc chloride (ZnCl₂) salt as reference. Through sequential screening over 24 h, our method identified 173 sensitive clones from diverse biological pathways, which fell into two general groups: early and late responders. The overlap between these groups was small. Our results suggest that bacterial toxicity mechanisms change from pathways related to general metabolic function, transport, signaling, and metal ion homeostasis to membrane synthesis pathways over time. While all zinc sources shared pathways relating to membrane damage and metal ion homeostasis, Zn-eNMs and ZnCl₂ displayed differences in their sensitivity profiles. For example, ZnCl₂ and nZnO elicited unique responses in pathways related to two-component signaling and monosaccharide biosynthesis, respectively. Single isolated measurements, such as MIC or IC₅₀, are inadequate, and time-resolved approaches utilizing genome-wide assays are therefore needed to capture this crucial dimension and illuminate the dynamic interplay at the nano-bio interface

Peptide substrates used for imaging probes preparation and their characterization.

<p>Peptide substrates used for imaging probes preparation and their characterization.</p

The probe chemical structure and action mechanism.

<p>(a) Chemical structures of optical probes. (b) Action mechanism of CTSE optical probe.</p

HE staining, immunohistochemical analysis and Western blot studies of CTSE of <i>in situ</i> DMBA-induced PDAC and PanINs.

<p>(a-e) Representative images of histology (HE staining) in pancreatic tissue sections of (a) normal pancreas; (b) PanIN-I; (c) PanIN-II; (d) PanIN-III; and (e) PDAC. (f-j) Representative images of immunohistochemical localization of CTSE in pancreas tissue sections of (f) normal pancreas; (g) PanIN-I; (h) PanIN-II; (i) PanIN-III; and (j) PDAC, showing a gradually increasing trend of CTSE expression (brown) with increasing grade of maglignancy. (k) Western blot shows very low levels in normal pancreatic tissues, but higher levels in PanINs and PDAC tumors. Scale bar, 100 µm.</p

Process of <i>in situ</i> DMBA-induced pancreatic adenocarcinoma in rats.

<p>Abbreviations: DMBA, 7,12-dimethyl-1,2-benzanthracene; PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma.</p><p>Process of <i>in situ</i> DMBA-induced pancreatic adenocarcinoma in rats.</p

<i>In vitro</i> imaging and quantitative determination of PDAC and PanINs in DMBA-induced rats injected with probe A.

<p>(a) <i>In vitro</i> imaging of tissue from tumors (R1 = PanIN-I; R2 = PanIN-II; R3 = PanIN-III; R4 = PDAC), kidney, liver, spleen, muscle and normal pancreas of four rats. (b) Quantitative analysis of near-infrared fluorescence imaging of tumor tissue and normal pancreatic tissue from four rats. (c-g) Routine pathological HE staining of (c) normal pancreas; (d) PanIN-I (R1); (e) PanIN-II (R2); (f) PanIN-III (R3); and (g) PDAC (R4). M = muscle; T = tumor; L = liver; S = spleen; K = kidney; P = pancreas. Scale bar, 100 µm.</p

<i>In vivo</i> CLE imaging of DMBA-induced PDAC in rats and pathological comparison.

<p>(a) <i>In vivo</i> CLE imaging of normal pancreas, low- and high-grade PanIN lesions, and PDAC (blue) with the activated CTSE-sensitive probe. (b) Representative images of histology in pancreas tissue sections from normal pancreas, low- and high-grade PanIN lesions, and PDAC. (c) Representative images of immunohistochemical localization of CSTE in pancreas tissue sections from normal pancreas, low- and high-grade PanIN lesions, and PDAC. Scale bar, 100 µm.</p