Demonstration of metastatic colon cancer cells adjacent to hepatic tissue.

<p>H&E, confocal and fluorescence images of adjacent tissue samples are shown (Confocal and fluorescence images from same slide). The tumor margin was much better delineated when employing fluorescence microscopy.</p

Liver tissue concentrations of the dye anti-CEA antibody conjugates over time.

<p>The liver was assayed to demonstrate tissue accumulation of the non-PEGylated and PEGylated 650 and 750 dyes that were conjugated to chimeric anti-CEA antibody. The non-PEGylated 650 (panel A) and 750 dye antibody conjugates (panel B) accumulated to significantly higher levels in the liver compared to their respective PEGylated dye antibody conjugates.</p

Characterization of pancreatic cancer cell lines.

<p>(A) Western blot analysis shows MUC1 expression in pancreatic cancer cell lines (BxPC-3 and Panc-1). (B) Flow cytometric analysis shows the expression of MUC1 on the surface of BxPC-3 and Panc-1 cell lines. (C) Immunocytochemistry on live cells shows multiple fluorescent dots on the surface of Panc-1 cells. Representative fluorescence images merged with corresponding DIC (differential interference contrast) images (x60 water immersion objective on FV1000, using the 559 nm laser).</p

Imaging of MUC1 targeting of orthotopically-transplanted Panc-1 and BxPC-3 pancreatic tumor in vivo.

<p>Fluorescence signals from pancreatic tumors orthotopically transplanted at the tail of the pancreas were detected. Other than the tumor, fluorescence signal was detected from the skin and, bladder and intestinal contents but at lower intensity than the tumor. White arrows indicate pancreatic tumor.</p

Imaging of MUC1 targeting of pancreatic tumors growing on skin flaps.

<p>Representative images were obtained under white light, and 473 nm and 559 nm lasers on the OV100, and merged. GFP signal from the individual cancer cells and red fluorescent signal from the DyLight 550 fluorophore-conjugated anti-MUC1 antibody at the outside margin of the colony and space between the cancer cells were observed.</p

Schematic comparison of anti-CEA chimeric antibodies with PEGylated and non-PEGylated NIR dyes.

<p>Four mPEG200 chains are covalently linked to each dye molecule, 4 of which are covalently linked to the chimeric anti-CEA antibody by amide bonds. The mPEG chains alter tissue biodistribution, allowing brighter liver metastases labeling and decreased accumulation in normal organs, particularly the liver.</p

Specificity of anti-CEA antibody conjugated dyes as compared to free dyes.

<p>In all images from A to D, the top panel of images is taken with the liver in anatomical position and the bottom panel is taken with the liver flipped upward to enable visualization of the undersurface. Image A is taken 24-anti-CEA antibody conjugate. Imaging at 750 nm clearly delineates all the HT-29-GFP liver metastases which are not completely visible on imaging with brightfield or GFP imaging (see arrows). Image B is taken 24 hours after injection of the 750 PEG dye only. No visible tumor labeling is noted, with little to no visualization of the dye throughout the animal, despite the GFP signal of the tumor being clearly detected. Image C is taken 24 hours after injection of the 650 PEG-anti-CEA antibody conjugate. There is very precise and clear labeling of tumor that is detected at 650 nm, which correlates well with the GFP signal. With the liver in the anatomical position, the gallbladder is also noted emerging from the underside of the liver. On the underside of the liver, a tumor is detected that is not visible on either GFP or brightfield imaging (see arrows). Image D is taken 24 hours after injection of the 650PEG free dye. There is non-specific distribution of the dye noted at 650 nm, with no selectivity for the tumor despite there being a robust GFP signal.</p

Whole body time sequence images of both PEGylated and non-PEGylated dye-anti-CEA antibody conjugates to evaluate total body biodistribution.

<p>After the mice are sacrificed at the indicated time-points, they were opened up from sternum to pubis to completely expose the viscera. In 2a, DyLight 650 anti-CEA antibody conjugate is evaluated, with images taken at different time-points. The top row is the PEGylated dye anti-CEA antibody conjugate, while the bottom row is the non-PEGylated anti-CEA antibody conjugate. With the PEGylated anti-CEA antibody conjugate, there is minimal to no accumulation of dye in the viscera, while in the non-PEGylated anti-CEA antibody conjugate, there is accumulation of dye in the reticulo-endothelial system organs such as the liver and lung. Arrows in the bottom row point to lymph nodes that retained and were labeled by the dye. In 2b, DyLight 750 is evaluated, with images taken at different time-points. The top row is the PEGylated dye anti-CEA antibody conjugate, while the bottom row is the non-PEGylated anti-CEA antibody conguate. With the PEGylated anti-CEA antibody conjugate, there was minimal to no accumulation of dye in the viscera, while in the non-PEGylated anti-CEA antibody conjugate there was accumulation of dye in the liver and lung. Arrows in the bottom row point to lymph nodes that retained and were labeled by the dye.</p

Fluorescence-Guided Surgery of Liver Metastasis in Orthotopic Nude-Mouse Models

<div><p>We report here the development of fluorescence-guided surgery of liver metastasis. HT29 human colon cancer cells expressing green fluorescent protein (GFP) were initially injected in the spleen of nude mice. Three weeks later, established liver metastases were harvested and implanted on the left lobe of the liver in other nude mice in order to make an orthotopic liver metastasis model. Fourteen mice with a single liver metastasis were randomized into bright-light surgery (BLS) or fluorescence-guided surgery (FGS) groups. Seven mice were treated with BLS, seven were treated with FGS. Three weeks after implantation, the left lobe of the liver with a single metastasis was exposed through a median abdominal incision. BLS was performed under white light. FGS was performed using a hand-held portable fluorescence imaging system (Dino-Lite). Post-surgical residual tumor fluorescence was visualized with the OV100 Small Animal Imaging System. Residual tumor fluorescence after BLS was clearly visualized at high magnification with the OV100. In contrast, residual tumor fluorescence after FGS was not detected even at high magnification with the OV100. These results demonstrate the feasibility of FGS for liver metastasis.</p></div

Pre-operative and post-operative images from the orthotopic liver metastasis model treated with FGS.

<p><b>(A)—(C)</b> Upper panels show bright field images, and lower panels are images of tumor fluorescence obtained with the OV100. Residual tumor fluorescence could not be detected even at high magnification <b>(C). (D,F,H)</b> Pre-FGS tumor fluorescence was clearly visualized with the Dino-Lite imaging system. <b>(E,G,I)</b> Dino-Lite imaging showed no evidence of tumor after FGS. <b>(J-K)</b> Dino-Lite settings. <b>(J)</b> After exposing the left lobe of the liver, the mouse was put under the Dino-Lite. <b>(K)</b> Connection between the Dino-Lite and computer. Tumor fluorescence was imaged on the monitor during FGS. Magnifications are indicated above the columns.</p