Imaging of primary and metastatic pancreatic cancer using a fluorophore-conjugated anti-CA19-9 antibody for surgical navigation

Abstract

Background: Despite recent surgical advances, pancreatic cancer remains the fourth leading cause of cancer-related death in the United States. This is due to inaccurate staging and difficulty in achieving negative margins at the time of pancreaticoduodenectomy. CA19-9 is a carbohydrate tumor-associated antigen found in up to 94% of pancreatic adenocarcinomas. In this study we investigate the use of a fluorophore-labeled anti-CA19-9 monoclonal antibody to improve intraoperative visualization of both primary and metastatic tumors in a mouse model of pancreatic cancer.

Methods: A monoclonal antibody specific for CA19-9 was conjugated to a green fluorophore and delivered to tumor-bearing mice as a single intravenous (IV) dose. Intravital fluorescence imaging was used to localize tumor implants 24 h after antibody administration.

Results: Using fluorescence imaging, the primary tumor was clearly visible at laparotomy, as were small metastatic implants within the liver and spleen and on the peritoneum. These tumor implants, which were nearly impossible to see using standard bright-field imaging, demonstrated clear fluorescence under LED light excitation. The fluorescence signal within the tumor tissue was maintained for over 3 weeks after a single administration of the labeled antibody. Histologic evaluation of tissue from animals treated with the conjugated anti-CA19-9 antibody likewise revealed strong staining of the tumor cells with minimal background staining of the peritumoral stroma.

Conclusions: Fluorophore-labeled anti-CA19-9 offers a novel intraoperative imaging technique for enhanced visualization of primary and metastatic tumors in pancreatic cancer when CA19-9 expression is present and may improve intraoperative staging and efficacy of resection.

Fig. 1

In vitro fluorescence intensity over time. The human pancreatic cancer cell line BxPC3 was plated at 10⁵ cells per well in a 96-well plate. Forty-eight hours later the confluent monolayer of cells was incubated with conjugated monoclonal CA19-9. The cells were then washed and imaged using an inverted fluorescence microscope at 100× magnification. Each day the cells were washed and the medium was replaced prior to imaging. Wells were evaluated in triplicate. The fluorescence signal remained present within the monolayer for 5 days

Fig. 2

CA19-9 dose-response. Athymic nude mice with small (1-2-mm diameter) flank tumors were given a single intravenous dose of CA19-9 ranging from 7.5 to 100 μg. All animals were imaged 24 h later and the fluorescence intensity of the tumors was measured using Image-J software. Tumor fluorescence was visible above background at 12.5 μg and all higher doses. Increasing antibody dose correlated with increasing fluorescence intensity across all doses given (n = 11)

Fig. 3

Time course of in vivo fluorescence intensity after intravenous delivery of conjugated CA19-9. Athymic nude mice with small (1-2-mm diameter) flank tumors were given a single intravenous dose of 75 μg of CA19-9. The animals were anesthetized and the tumors were imaged via a skin flap 1, 2, 3, 5, 7, 9, 11, 13, 15, 19, and 23 days after antibody delivery. The fluorescence signal remained present for over 3 weeks after antibody administration (n = 3)

Fig. 4

Intravital imaging of pancreatic cancer after intravenous delivery of conjugated CA19-9. Two weeks after surgical orthotopic implantation of solid tumor to the pancreas, mice were given a single intravenous dose of 75 μg of CA19-9. The animals were anesthetized 24 h later and the tumors within the pancreas were imaged at laparotomy. Small tumors that were indistinguishable from normal adjacent pancreas under brightfield (A) were clearly visible using fluorescence (C). Control animals bearing orthotopic tumors without exposure to conjugated anti-CA19-9 (B) showed low autofluorescence of the primary tumor under fluorescence (D) (n = 4)

Fig. 5

H&E and fluorescence imaging of sectioned pancreatic tumors after intravenous delivery of conjugated CA19-9. Following antibody administration, laparotomy, and intravital imaging of orthotopic tumors, the pancreatic tumors were resected en bloc with surrounding normal pancreatic tissue and frozen in OCT for sectioning. Thick (15 mm) sections were evaluated unfixed for green fluorescence and thin (8 mm) sections were prepared by H&E staining. Evaluation of H&E-stained thin sections under standard light microscopy at 200× magnification revealed a characteristic pattern of clusters of tumor cells surrounded by a dense network of stromal cells in all tumors sectioned (B, D). Fluorescence microscopy of unfixed thick sections under 200× magnification revealed a bright green signal from the tumor cells with minimal staining of the surrounding peritumoral stroma (A), while a sectioned tumor from a control animal that was not given antibody revealed essentially no fluorescence (C) (n = 4)

Fig. 6

Experimental metastatic splenic lesions imaged after intravenous delivery of conjugated CA19-9. Approximately 1 week after direct injection of tumor cells to the spleen, the mice were given a single intravenous dose of 75 μg of CA19-9. Twenty-four hours later the animals were anesthetized and evaluated at lapaotomy via intravital imaging using both bright-field and fluorescence imaging. In the spleen, micrometastases, which were indistinguishable from normal tissue under bright-field imaging, even at high magnification (A-C), were clearly visible using fluorescence imaging (D-F)

Fig. 7

Metastatic liver lesions imaged after intravenous delivery of conjugated CA19-9. Approximately 2 weeks after implantation of tumor cells to the liver, the mice were given a single intravenous dose of 75 μg of CA19-9. Twenty-four hours later the animals were anesthetized and evaluated at laparotomy using both standard white-light illumination and fluorescence imaging. Micrometastases that were indistinguishable from normal tissue under bright-field imaging, even at high magnification (A-C), were clearly visible using fluorescence imaging (D-F)

Fig. 8

Peritoneal tumor implants imaged after intravenous delivery of conjugated CA19-9. Approximately 1 week after injection of tumor cells into the abdominal cavity, mice were given a single intravenous dose of 75 μg of CA19-9. Twenty-four hours later the animals were anesthetized and evaluated at laparotomy using both brightfield and fluorescence imaging. Small peritoneal implants that could not be observed, even at the highest magnifications under bright-field imaging (A), were visible when imaged with an LED light source after administration of conjugated antibody (B). Animals with larger peritoneal implants that did not receive the antibody (C) showed minimal autofluorescence of the tumor implants under fluorescence imaging (D)