Volume 138, Issue 2, Pages 435-446 (February 2010) In Vivo Molecular Imaging of Colorectal Cancer With Confocal Endomicroscopy by Targeting Epidermal Growth Factor Receptor  Martin Goetz, Alex Ziebart, Sebastian Foersch, Michael Vieth, Maximilian J. Waldner, Peter Delaney, Peter R. Galle, Markus F. Neurath, Ralf Kiesslich  Gastroenterology  Volume 138, Issue 2, Pages 435-446 (February 2010) DOI: 10.1053/j.gastro.2009.10.032 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 EGFR staining ex vivo. After FITC-labeling of tumor cells with anti-EGFR antibody in vitro, FACS analysis clearly differentiates between SW480 cells (A) with a high EGFR expression and SW620 cells with a low EGFR expression (B). These expression patterns are confirmed by IHC of cytospins (C and D). Gastroenterology 2010 138, 435-446DOI: (10.1053/j.gastro.2009.10.032) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Molecular imaging of cell cultures by CLE. Unfixed SW480 cells (upper panels) or SW620 cells (lower panels) were incubated with labeled EGFR antibody and examined by handheld confocal microscopy. An intravital signal was observed in EGFR-positive SW480 cells (arrows) but not in EGFR-negative SW620 cells with CLE (A and D). With the same staining protocol, bench-top fluorescence microscopy confirmed these expression patterns, displaying bound antibody in green on SW480 cells (B) but not on SW620 cells (E). Overlay with nuclear counterstaining (DAPI blue) helped to localize EGFR fluorescence to superficial or cytoplasmic cellular compartments (C and F). Gastroenterology 2010 138, 435-446DOI: (10.1053/j.gastro.2009.10.032) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 High-resolution molecular imaging in vivo. Within a SW480 tumor, a membranous staining pattern is visualized in vivo (arrows, A). Magnification of a single cell (boxed area in A) shows accumulation of the fluorescence signal at the level of the cell surface (arrows, B). Gastroenterology 2010 138, 435-446DOI: (10.1053/j.gastro.2009.10.032) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Molecular imaging of SW480 tumors in live mice. CLE showed a clear specific signal within the tumor tissue after systemic application of FITC-labeled anti-EGFR antibody (arrows, A). Specificity of this signal was confirmed by ex vivo IHC of tumor sections (arrows, B). Ex vivo bench-top fluorescence microscopy confirmed the FITC signal (arrows, green) within the tumor after intravital staining (C; overlay with ex vivo nuclear counterstaining [blue], D). Gastroenterology 2010 138, 435-446DOI: (10.1053/j.gastro.2009.10.032) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Molecular imaging of SW620 tumors in live mice. No specific signal was observed in a typical SW620 tumor after the injection of FITC-labeled anti-EGFR antibody by CLE (A), IHC (B), or bench-top fluorescence microscopy (C and D). Gastroenterology 2010 138, 435-446DOI: (10.1053/j.gastro.2009.10.032) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Molecular imaging of an EGFR-positive liver metastasis. To mimic a portal metastasis route, SW480 cells were injected into the spleen. After injection of FITC-labeled EGFR antibody, the invasion front of a liver metastasis was delineated against the unstained normal parenchyma (A). A targeted biopsy (H&E stain) showed the tumor cells invading the healthy liver tissue (B). Detailed inspection (insets) revealed the cytoplasmic fluorescence pattern of this EGFR-positive metastasis with a central clearance (C), in which the enlarged nuclei are located, as demonstrated by ex vivo H&E staining (D). Gastroenterology 2010 138, 435-446DOI: (10.1053/j.gastro.2009.10.032) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Quantification of specific in vivo fluorescence. Signal strength was graded from absent (0) to weak (+), moderate (++), and strong (+++) (A). A significantly stronger expression was demonstrated in SW480 tumors (light columns) than in SW620 tumors (dark columns), P < .01 (B). Mean specific fluorescence in vivo after EGFR labeling was 0.59 ± 0.21 in SW620 tumors and 1.92 ± 0.22 in SW480 tumors (P = .0004). Bars indicate SEM (C). Gastroenterology 2010 138, 435-446DOI: (10.1053/j.gastro.2009.10.032) Copyright © 2010 AGA Institute Terms and Conditions

Figure 8 Molecular imaging in humans. Tissue samples from a neoplastic lesion were incubated in labeled EGFR antibody solution to mimic topical application during colonoscopy (A). CLE showed a specific signal within the lesion (arrows, B). Mean specific fluorescence of human tissue samples was 0.25 ± 0.16 for normal mucosa vs 2.12 ± 0.30 for neoplasia (P < .002, C). Bars indicate SEM. Histopathology confirmed malignancy, and IHC showed EGFR expression in multiple tumor cells (arrows, D). Gastroenterology 2010 138, 435-446DOI: (10.1053/j.gastro.2009.10.032) Copyright © 2010 AGA Institute Terms and Conditions