In vivo spectral fluorescence imaging of submillimeter peritoneal cancer implants using a lectin-targeted optical agent

Abstract

Intraperitoneal metastases commonly recur after surgery because small tumor foci escape detection within the complex anatomy of the peritoneal cavity and mesentery. Accurate localization of peritoneal implants during surgery could improve the resection of ovarian cancer and other malignancies, but few practical techniques to enhance detectability are currently available. Here, we describe a targeted molecular imaging method that employs fluorescently labeled avidin to detect submillimeter peritoneal implants of ovarian cancer in mice. After binding to surface lectins on the tumor, fluorescein-conjugated avidin enabled the spectral fluorescence imaging of disseminated peritoneal implants. High spatial resolution and high tumor-to-background ratio allowed the visualization of implants as small as 0.3 mm (with 100% sensitivity and specificity; n = 150) and the identification of even smaller lesions ex vivo. These results suggest that targeted molecular imaging with a fluorescence-labeled lectin-ligand system is a promising technique for the detection of disseminated submillimeter foci of cancer.

Figure 1

In vivo spectral fluorescence images of tumor-bearing mice. Composite images (red: autofluorescence; green: FITC fluorescence) were obtained 1, 2, 4, and 8 hours after injection of 50 µg of Avidin-FITC (A, 1 hour; B, 2 hours; C, 4 hours; D, 8 hours). (A and B) High fluorescence intensity was observed in tumor foci, but the background signal was still high within 2 hours postinjection. (C) Aggregated tumor foci (white arrowheads) and a small tumor nodule (white arrow) measuring < 5 mm are clearly shown 4 hours postinjection. (D) At 8 hours postinjection, the fluorescence from tumor foci (blue arrowheads) became very weak and almost unidentifiable. Imaging is optimized at approximately 4 hours postinjection.

Figure 2

Sensitivity, specificity, and detection limit. (A) Ex vivo composite image of tumor and organ. Four hours postinjection, the image clearly distinguished the Avidin-FITC fluorescence of the tumor from the autofluorescence of the gastrointestinal tracts and of other organs (red: autofluorescence; green: FITC fluorescence). The arrowhead indicates the gallbladder. T, tumor nodules; Li, liver; Sp, spleen; St, stomach; I, intestine; K, kidneys; Lu, lung; H, heart. (B) Microscopy of extracted peritoneal implants. SHIN3 cancer implants (arrows) were located on the free surface of the peritoneal cavity (original magnification, x200; H&E stain). (C) In vivo composite image of the peritoneum (red: autofluorescence; green: FITC fluorescence) obtained 4 hours postinjection of 50 µg of Avidin-FITC clearly depicted submillimeter peritoneally disseminated implants of ovarian cancer. Cancer implants as small as 0.3 mm are clearly depicted. The scale on the ruler represents 1 mm. (D) High-resolution fluorescence image of peritoneal implants. High fluorescence signal can be seen to a depth of approximately 100 µm along the peritoneal surface of the tumor (arrowheads).

Figure 3

Nonspectral fluorescence images of extracted peritoneal membrane with (A, B, D) or without (C) tumor implants. (A) Fluorescence image of NeutrAvidin-FITC at a dose of 65 µg. Sixty-five micrograms of NeutrAvidin-FITC contains the same number of FITC molecules as 50 µg of Avidin-FITC. Implants are invisible. (B) Fluorescence image of unconjugated FITC at a dose of 0.73 µg. An amount of 0.73 µg of FITC contains the same number of FITC molecules as 50 µg of Avidin-FITC. Implants and vascular structures are barely seen. (C) Fluorescence image of Avidin-FITC at a dose of 50 µg in a normal mouse without tumor. No focal uptake is shown. (D) Fluorescence image of Avidin-FITC at a dose of 50 µg. Relatively larger tumor foci (> 1 mm in diameter) are easily detectable (arrows), whereas smaller foci are barely visible (arrowheads). The ruler represents 1 mm. The exposure time was 500 milliseconds in all images.

Figure 4

In vivo and in vitro fluorescence microscopy. Avidin-FITC similarly accumulated in SHIN3 cells both in vivo and in vitro. (A) Fluorescence microscopy of the peritoneal implants 4 hours postinjection with 50 µg of Avidin-FITC demonstrates clusters of punctate fluorescence within the cells (original magnification, x200). In vitro fluorescence microscopy (B) and DIC (C) images of SHIN3 cancer cells. After 4 hours of incubation with Avidin-FITC (333 ng/ml), a large number of fluorescent dots are detected within the cytoplasm of SHIN3 cells.