Introduction: The use of near infrared (NIR) fluorescence imaging technology in surgical navigation has demonstrated significant potential. Antibody-fluorophore conjugates show promise in labeling tumors in-situ in real-time, with many progressing in clinical trials. However, most current fluorescence imaging systems detect fluorescence intensity, which cannot delineate non-specific accumulation of the probe in off-target locations from tumor specific probe, thereby increasing false-positives. Fluorescence lifetime (FLT) imaging has emerged as a valuable adjunct since cancers exhibit altered fluorescence lifetimes in comparison to normal tissue¹. Here, we evaluate FLT based contrast enhancement of an antibody fluorophore conjugate that binds to the human carcinoembryonic antigen in an orthotopic mouse model of colorectal cancer.

Materials/ Methods: The humanized anti-CEA hT84.66-M5A (M5A) or a nonspecific IgG monoclonal antibody (mAb) were conjugated to LICOR-IRDye800CW using NHS chemistry to create the M5A-IR800CW or IgG-IR800CW conjugates². Orthotopic mouse models of colorectal cancer were established by surgical implantation of LS174T human colorectal cancer tumor fragments into the cecum of nude mice. Mice received 75 ug of either IgG-IR800CW or M5A-IR800CW conjugates intravenously. Mice were imaged at 4 hours and 48 hours. NIR fluorescence imaging was performed using the LICOR-Pearl. Tumor, adjacent organs, and liver were harvested. Fixed and paraffin-embedded specimen blocks were created. Tissue blocks underwent widefield imaging using white light, NIR continuous wave fluorescence, and time domain (FLT) imaging (Kumar lab, proprietary imaging device). Fixed and paraffin-embedded specimen were sectioned into 10 µm sections and imaged using the Stellaris 8 Falcon (Leica, Germany) fluorescence lifetime microscope system with 730 nm excitation and a 750 nm notch filter; emission was detection with a HyD R detector at 770-850 nm. Time domain data was collected using time-correlated single photon counting.

Results: There was limited contrast over the tumor using M5A-IR800CW and IgG-IR800CW conjugates using both continuous wave and fluorescence lifetime imaging. Near-infrared widefield continuous wave imaging of orthotopic colorectal tumors demonstrated a strong fluorescence signal from the tumors and the liver using M5A-IR800CW (Fig 1A). Widefield FLT imaging demonstrated a longer fluorescence lifetime over the tumor (1.1973 ns) as compared to the liver (0.8929 ns) (Fig 1B, middle). The tumor and liver were not distinguishable from corresponding continuous wave fluorescence image of the same specimen, which showed comparable fluorescence signal from both liver and tumor (Figure 1B, right). The FLT contrast seen in the blocks was confirmed using fluorescence lifetime microscopy (Fig 1C, middle) of tissue sections. On standard fluorescence microscopy, there was comparable fluorescence signal over both the liver and the tumor sections (Fig 1C, right). A fluorescence signal was not detectable over the tumor or the liver using IgG-IR800CW (Fig 2A). Widefield FLT imaging demonstrated similar fluorescence lifetimes over the tumor (0.5116 ns) and the liver (0.5044 ns) (Fig 2B). A similar fluorescence lifetime between the tumor and the liver was observed on fluorescence lifetime microscopy (Fig 2C, middle), in contrast to the M5A which showed a significant FLT contrast. On standard fluorescence microscopy, there was a fluorescence signal over both the liver and the tumor sections (Fig 2C, right). Conclusions: The use fluorescence lifetime imaging can further augment the accuracy of tumor-targeted fluorescent probes³. In this work, the humanized anti-CEA antibody labeled with IRDye800CW was evaluated. FLT imaging was able to distinguish between a tumor-specific signal and the non-specific liver accumulation of antibody-fluorophore conjugates. This technology demonstrates fidelity at the wide-field and microscopic scale. Furthermore, FLT can allow signal comparison across different agents and imaging systems, overcoming the limitations of continuous wave fluorescence imaging due to excitation source, imaging distance, and system-specific measurement parameters. The work demonstrates a highly promising potential application of FLT for next-generation fluorescence-guided surgery.

Image/Figure Caption:

Near-infrared widefield continuous wave imaging of orthotopic colorectal tumors demonstrated a strong fluorescence signal from the tumors and the liver using M5A-IR800CW (Fig 1A). Widefield FLT imaging demonstrated a longer fluorescence lifetime over the tumor (1.1973 ns) as compared to the liver (0.8929 ns) (Fig 1B, middle). The tumor and liver were not distinguishable from corresponding continuous wave fluorescence image of the same specimen, which showed comparable fluorescence signal from both liver and tumor (Figure 1B, right). The FLT contrast seen in the blocks was confirmed using fluorescence lifetime microscopy (Fig 1C, middle) of tissue sections. On standard fluorescence microscopy, there was comparable fluorescence signal over both the liver and the tumor sections (Fig 1C, right). A fluorescence signal was not detectable over the tumor or the liver using IgG-IR800CW (Fig 2A). Widefield FLT imaging demonstrated similar fluorescence lifetimes over the tumor (0.5116 ns) and the liver (0.5044 ns) (Fig 2B). A similar fluorescence lifetime between the tumor and the liver was observed on fluorescence lifetime microscopy (Fig 2C, middle), in contrast to the M5A which showed a significant FLT contrast. On standard fluorescence microscopy, there was a fluorescence signal over both the liver and the tumor sections (Fig 2C, right).

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