@inproceedings{01HTDMCY2PJTF40YVVGZ2NFJM8,
  abstract     = {{Introduction

Preclinical in vivo and clinical ex vivo studies suggest that fluorescence lifetime (FLT) imaging has higher sensitivity and specificity for tumor identification compared to intensity-based imaging, as FLT imaging provides additional information on the environment of the fluorophore. Towards future implementation of FLT imaging in the context of image-guided surgery, preclinical FLT studies were conducted with a novel FLT imaging system, the tauCAM. Here, we present how FLT imaging could aid in more specific tumor imaging, and the importance of fluorophore selection.

Methods

Mice (n=3 per group) bearing a subcutaneous EGFR+ tumor were intravenously injected with the anti-EGFR mAb cetuximab or the smaller anti-EGFR Nanobody (Nb) 7D12 conjugated to either IRDye800CW or s775z. Longitudinal imaging was performed up to 48 hours post-injection (p.i.) using the tauCAM, a custom-built 128x128-pixel CMOS time-gated camera that can efficiently determine the FLT of near-infrared fluorescence. After euthanasia, ex vivo imaging of dissected organs was conducted to confirm the in vivo findings. In addition, conditions triggering FLT differences were investigated in vitro using 2D and 3D cell cultures using FLT imaging microscopy with a Leica Stellaris 8 Falcon microscope.

Results/Discussion

On intensity images, all tracers showed tumor accumulation reaching a peak at 48h and 1h, respectively, for the EGFR-mAbs and -Nbs. However, background signals were also seen in the liver for the mAbs and in the kidneys for the Nbs. Based on FLT imaging, a clear difference between cetuximab-IRDye800CW in the tumor and background tissues could be observed over time. For Nb-IRDye800CW, tumor and kidney signals could already be differentiated based on FLT measurements at 1h p.i. Contrarily, the FLT of the s775z-labeled analogues remained similar in all organs and tissues over time. These results were confirmed ex vivo. Finally, microscopic spheroid and 2D cell studies revealed that the FLT of IRDye800CW-tracers changes after endocytosis, whereas that of s775z-tracers does not.

Conclusion

We demonstrate that through the use of fluorophores, sensitive to their physiological environment, FLT imaging has an advantage over intensity-based imaging to differentiate specific tumor uptake from non-specific background signals. Moreover, the combination of FLT with fluorophores which are less or not responsive to their environment, such as s775z, could be useful for the simultaneous imaging of multiple fluorophores in the same spectrum.

Novelty

Real-time FLT imaging in NIR is a emerging field.This study demonstrates the importance of the properties of the fluorophore for FLT imaging.

Impact

Effective real-time FLT imaging could have high potential for improved tumor imaging during fluorescence-guided surgery.

Disclosure

VUB is patent owner of a patent on the image sensor used in the FLT camera (inventor: Hans Ingelberts)

Acknowledgment
Funding provided by Fonds voor Wetenschappelijk onderzoek (FWO), project "NLITESurgery"}},
  author       = {{Janssen, Simone and Van den Dries, Thomas and Stroet, Marcus and Sahakian, Sevada and Lapauw, Thomas and Okkelman, Irina and Dmitriev, Ruslan and Kuijk, Maarten and Ingelberts, Hans and Hernot, Sophie}},
  booktitle    = {{19th European Molecular Imaging Meeting (EMIM 2024), Abstracts}},
  language     = {{eng}},
  location     = {{Porto, Portugal}},
  title        = {{In Vivo fluorescence lifetime imaging : added value for tumor imaging and importance of fluorophore selection}},
  url          = {{https://www.eventclass.it/emim2024/scientific/online-program/poster-session?s=PO+II}},
  year         = {{2024}},
}