Advantages of fluorescence-guided laparoscopic surgery of pancreatic cancer labeled with fluorescent anti-carcinoembryonic antigen antibodies in an orthotopic mouse model

Abstract

Background: Our laboratory has previously developed fluorescence-guided surgery of pancreatic and other cancers in orthotopic mouse models. Laparoscopic surgery is being used more extensively in surgical oncology. This report describes the efficacy of laparoscopic fluorescence-guided surgery of pancreatic cancer in an orthotopic mouse model.

Study design: Mouse models of human pancreatic cancer were established with fragments of the BxPC-3 red fluorescent protein-expressing human pancreatic cancer using surgical orthotopic implantation. Mice were randomized to bright-light laparoscopic surgery (BLLS) or to fluorescence-guided laparoscopic surgery (FGLS). Fluorescence-guided laparoscopic surgery was performed with a light-emitting diode light source through a 495-nm emission filter in order to resect the primary tumors and any additional separate submillimeter tumor deposits within the pancreas, the latter of which was not possible with BLLS. Tumors were labeled with anti-CEA AlexaFluor 488 antibodies 24 hours before surgery with intravenous injection. Perioperative fluorescence images were obtained to evaluate tumor size. Mice were followed postoperatively to assess for recurrence and at termination to evaluate tumor burden.

Results: At termination, the FGLS-treated mice had less pancreatic tumor volume than the BLLS-treated mice (5.75 mm(2) vs 28.43 mm(2), respectively; p = 0.012) and lower tumor weight (21.1 mg vs 174.4 mg, respectively; p = 0.033). Fluorescence-guided laparoscopic surgery compared with BLLS also decreased local recurrence (50% vs 80%, respectively; p = 0.048) and distant recurrence (70% vs 95%, respectively; p = 0.046). More mice in the FGLS group than the BLLS group were free of tumor at termination (25% vs 5%, respectively). Median disease-free survival was lengthened from 2 weeks with BLLS (95% CI, 1.635-2.365) to 7 weeks with FGLS (95% CI, 5.955-8.045; p = 0.001).

Conclusions: Fluorescence-guided laparoscopic surgery is more effective than BLLS and, therefore, has important potential for surgical oncology.

Copyright © 2014 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Labeling Efficacy of Chimeric Anti-CEA-Alexa 488 Antibodies

A representative bright field image of a resected pancreatic tumor is show in panel (a). The RFP-expressing tumor is visualized under the RFP filter (excitation 535-555, emission 570-623) of the OV-100 Small Animal Imaging System (b). With a GFP filter (excitation 460-490, emission 510-550) (c), only the tumor labeled with the green fluorescent antibody is visualized. The accuracy of the green fluorescent antibody in labeling the CEA-expressing RFP pancreatic tumor results in a yellow image under the GFP filter (excitation 460-490, emission 510F), which can visualize both green and red fluorescence (d).

Figure 2. FGLS vs BLLS for pancreatic cancer

a) The RFP-expressing pancreatic tumor (seen in the FL image) is difficult to identify under standard bright lighting (BLL). However, when the tumor was labeled with the chimeric anti-CEA-488 antibody, identification and visualization of the tumor was significantly enhanced allowing for better resection under fluorescence-guidance (bottom images). BLL, bright light laparoscopy; FL, fluorescence laparoscopy; FGLS, fluorescence-guided laparoscopic surgery; RFP, red fluorescent protein. b) Representative postoperative images from the BLLS group and the FGLS group. The arrow identifies residual red fluorescence on postoperative whole body images indicating an incomplete resection. This occurred in two out of 22 mice in the BLLS group. All 24 mice in the FGLS group underwent a complete resection. The preoperative image is representative of all mice undergoing resection. These images were obtained in order to confirm presence of tumor.

Figure 2. FGLS vs BLLS for pancreatic cancer

a) The RFP-expressing pancreatic tumor (seen in the FL image) is difficult to identify under standard bright lighting (BLL). However, when the tumor was labeled with the chimeric anti-CEA-488 antibody, identification and visualization of the tumor was significantly enhanced allowing for better resection under fluorescence-guidance (bottom images). BLL, bright light laparoscopy; FL, fluorescence laparoscopy; FGLS, fluorescence-guided laparoscopic surgery; RFP, red fluorescent protein. b) Representative postoperative images from the BLLS group and the FGLS group. The arrow identifies residual red fluorescence on postoperative whole body images indicating an incomplete resection. This occurred in two out of 22 mice in the BLLS group. All 24 mice in the FGLS group underwent a complete resection. The preoperative image is representative of all mice undergoing resection. These images were obtained in order to confirm presence of tumor.

Figure 3. Tumor burden at termination using BLLS and FGLS

There were no significant differences with regard to average preoperative tumor burden (p=0.657) or average resected specimen size (p=0.213) between the two surgical groups. However, the improved resection achieved under fluorescence-guidance led to significantly lower pancreatic tumor burden at termination in the FGLS group compared to the BLLS group (p=0.012).

Figure 4. Kaplan meier curve for disease-free survival with BLLS vs FGLS

FGLS improved DFS in mice harboring pancreatic tumor by more than doubling the average time in weeks compared to BLLS. Median DFS improved from 2 weeks in the BLLS group to 7 weeks, p=0.001.

Figure 5. Metastatic tumor burden at termination twelve weeks postoperatively with BLLS vs FGLS

Representative intravital images obtained with the OV-100 Small Animal Imaging System demonstrating improved outcomes achieved by FGLS compared to BLLS at termination. FGLS significantly reduced the number of mice with local and distant recurrence compared to BLLS.