Successful fluorescence-guided surgery on human colon cancer patient-derived orthotopic xenograft mouse models using a fluorophore-conjugated anti-CEA antibody and a portable imaging system

Abstract

Background: Fluorescence-guided surgery (FGS) can enable successful cancer surgery where bright-light surgery often cannot. There are three important issues for FGS going forward toward the clinic: (a) proper tumor labeling, (b) a simple portable imaging system for the operating room, and (c) patient-like mouse models in which to develop the technology. The present report addresses all three.

Materials and methods: Patient colon tumors were initially established subcutaneously in nonobese diabetic (NOD)/severe combined immune deficiency (SCID) mice immediately after surgery. The tumors were then harvested from NOD/SCID mice and passed orthotopically in nude mice to make patient-derived orthotopic xenograft (PDOX) models. Eight weeks after orthotopic implantation, a monoclonal anti-carcinoembryonic antigen (CEA) antibody conjugated with AlexaFluor 488 (Molecular Probes Inc., Eugene, OR) was delivered to the PDOX models as a single intravenous dose 24 hours before laparotomy. A hand-held portable fluorescence imaging device was used.

Results: The primary tumor was clearly visible at laparotomy with the portable fluorescence imaging system. Frozen section microscopy of the resected specimen demonstrated that the anti-CEA antibody selectively labeled cancer cells in the colon cancer PDOX. The tumor was completely resected under fluorescence navigation. Histologic evaluation of the resected specimen demonstrated that cancer cells were not present in the margins, indicating successful tumor resection. The FGS animals remained tumor free for over 6 months.

Conclusions: The results of the present report indicate that FGS using a fluorophore-conjugated anti-CEA antibody and portable imaging system improves efficacy of resection for CEA-positive colorectal cancer. These data provide the basis for clinical trials.

FIG. 1.

Representative images of the colon cancer patient-derived orthotopic xenograft (PDOX) sections. (A) Hematoxylin and eosin staining of a patient-derived orthotopic xenograft section. The tumor was diagnosed as moderately differentiated adenocarcinoma. (B) Immunohistochemistry for human carcinoembryonic antigen on a sister slide of A. High-intensity brown staining was observed in the cancer cells. Scale bars=250 μm.

FIG. 2.

Fluorescence-guided surgery of the primary colon tumor was performed using the portable imaging system with (A) the Dino-Lite digital camera. Eight weeks after orthotopic implantation of a colon cancer patient-derived orthotopic xenograft (PDOX), a monoclonal anti-carcinoembryonic antigen antibody conjugated with AlexaFluor^® 488 was delivered to tumor-bearing mice as a single intravenous dose. After 24 hours, fluorescence-guided surgery of the primary colon tumor was performed using the portable imaging system (B).

FIG. 3.

The colon cancer patient-derived orthotopic xenograft (PDOX) was imaged with the portable imaging system (A and B) or the Olympus OV100 small animal imaging system (C and D). During fluorescence-guided surgery, the tumor was clearly visible (A). The portable imaging system had less autofluorescence signal compared with the OV100 system. Scale bars=2 mm. BF, bright-field.

FIG. 4.

Frozen section microscopy of the resected specimen. (A) Hematoxylin and eosin staining of the resected specimen. (B) 4′,6-Diamidino-2-phenylindole staining of the resected specimen. Arrowheads indicate the border of the tumor. Tumor cells were clearly stained by anti-carcinoembryonic antigen antibody administered by intravenous injection 24 hours before laparotomy. Blue, 4′,6-diamidino-2-phenylindole; green, anti-carcinoembryonic antigen antibody conjugated with AlexaFluor^® 488. The sections were observed with confocal microscopy (Fluoview FV1000; Olympus). Scale bar=50 μm.

FIG. 5.

Tumor-bearing mice were imaged (A) before and (B) after resection. Lower panels are high magnification (×0.56) images of the upper panels. A complete absence of tumor was detected in the postsurgical bed. The images were taken with the Olympus OV100 Small Animal Imaging System under both standard bright-field (BF) and fluorescence illumination. Scale bars=10 mm in upper panels and 2 mm in lower panels.

FIG. 6.

Histological analysis of the margin of the resected specimen. (A) The box indicates the margin. (B) Hematoxylin and eosin staining of the margin. Cancer cells were not detected in the margin. Scale bars=2 mm in (A) and 250 μm in (B).