Confocal Imaging and Tissue-Specific Fluorescent Probes for Real-Time In Vivo Immunohistochemistry. Proof of the Concept in a Gastric Lymph Node Metastasis Model.
Tumor-specific fluorescent antibodies, which can be recognized at a cellular or tissue level using optical imaging such as confocal laser endomicroscopy (CLE), could provide a means for rapid and accurate tumor diagnosis and staging. The aim of this study was to evaluate the ability of CLE to detect the presence of tagged cells within lymph nodes in an original simulated metastatic model.



A solution of indocyanine green containing a suspension of porcine hepatocytes, marked with carboxy-fluorescein-succinimidyl-ester (CFSE), was injected endoscopically in the gastric submucosa of 10 pigs. Fluorescence lymphography using a near-infrared laparoscope was used to identify sentinel and secondary drainage nodes. Additionally, a nonfluorescent gastric and a mesenteric node were identified. Every 5-10 min, those nodes were scanned using probe-based or needle-based CLE (pCLE or nCLE). Immunohistochemistry (IHC) using anti-cytokeratin 18 antibodies was subsequently performed to confirm the presence of hepatocytes in the lymph nodes.



A total of 36 lymph nodes were analyzed with both CLE probes. Hepatocyte penetration in lymph nodes, as assessed by repeated CLE scanning, took 10-40 min after submucosal injection. Concordance between CLE and IHC was 84 and 72 % for pCLE and nCLE, respectively. False negatives were partly due to incomplete CFSE labeling of hepatocytes, which could not be recognized by CLE, but were detected with IHC.



Real-time CLE analysis effectively recognized the presence in perigastric nodes of marked hepatic cells that had been injected endoscopically in the stomach. Validation studies on tumor-bearing animals using tumor-specific antibodies should be performed.
Research Institute Against Cancer of the Digestive System, IRCAD, Strasbourg, France. michelediana@virgilio.it.|Institute of Image-Guided Surgery, IHU-Strasbourg, Strasbourg, France. michelediana@virgilio.it.
Michele
M
Diana
Institute of Image-Guided Surgery, IHU-Strasbourg, Strasbourg, France.
Eric
E
Robinet
Research Institute Against Cancer of the Digestive System, IRCAD, Strasbourg, France.|Department of General Surgery, Chang Gung Memorial Hospital, Linkou, Chang Gung University, Taoyuan, Taiwan.
Yu-Yin
YY
Liu
Institute of Image-Guided Surgery, IHU-Strasbourg, Strasbourg, France.
Andras
A
Legnèr
Institute of Image-Guided Surgery, IHU-Strasbourg, Strasbourg, France.|Department of Surgery, Seoul National University Hospital, Seoul, Korea.
Seong-Ho
SH
Kong
Research Institute Against Cancer of the Digestive System, IRCAD, Strasbourg, France.
Luigi
L
Schiraldi
Research Institute Against Cancer of the Digestive System, IRCAD, Strasbourg, France.
Francesco
F
Marchegiani
Institute of Image-Guided Surgery, IHU-Strasbourg, Strasbourg, France.
Peter
P
Halvax
Institute of Image-Guided Surgery, IHU-Strasbourg, Strasbourg, France.
Lee
L
Swanstrom
Research Institute Against Cancer of the Digestive System, IRCAD, Strasbourg, France.
Bernard
B
Dallemagne
Research Institute Against Cancer of the Digestive System, IRCAD, Strasbourg, France.|Institute of Image-Guided Surgery, IHU-Strasbourg, Strasbourg, France.
Jacques
J
Marescaux
D000818:Animals; D000906:Antibodies; D004396:Coloring Agents; D005260:Female; D005753:Gastric Mucosa; D022781:Hepatocytes / Q000276:immunology; D007150:Immunohistochemistry / Q000379:methods*; D007208:Indocyanine Green; D053538:Keratin-18 / Q000276:immunology; D010535:Laparoscopy / Q000379:methods; D008207:Lymphatic Metastasis; D008297:Male; D008643:Mesentery; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D000075082:Proof of Concept Study; D000071036:Sentinel Lymph Node / Q000000981:diagnostic imaging* / Q000473:pathology*; D013552:Swine
D000906:Antibodies; D004396:Coloring Agents; D053538:Keratin-18; D007208:Indocyanine Green