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. 2020 Apr 17;12(4):987.
doi: 10.3390/cancers12040987.

Ex Vivo Assessment of Tumor-Targeting Fluorescent Tracers for Image-Guided Surgery

Fortuné M K Elekonawo  1   2 , Jan Marie de Gooyer  1   2 , Desirée L Bos  1 , David M Goldenberg  3 , Otto C Boerman  2 , Lodewijk A A Brosens  4 , Andreas J A Bremers  2 , Johannes H W de Wilt  2 , Mark Rijpkema  1
Affiliations

Ex Vivo Assessment of Tumor-Targeting Fluorescent Tracers for Image-Guided Surgery

Fortuné M K Elekonawo et al. Cancers (Basel). .

Abstract

Image-guided surgery can aid in achieving complete tumor resection. The development and assessment of tumor-targeted imaging probes for near-infrared fluorescence image-guided surgery relies mainly on preclinical models, but the translation to clinical use remains challenging. In the current study, we introduce and evaluate the application of a dual-labelled tumor-targeting antibody for ex vivo incubation of freshly resected human tumor specimens and assessed the tumor-to-adjacent tissue ratio of the detectable signals. Immediately after surgical resection, peritoneal tumors of colorectal origin were placed in cold medium. Subsequently, tumors were incubated with 111In-DOTA-hMN-14-IRDye800CW, an anti-carcinoembryonic antigen (CEA) antibody with a fluorescent and radioactive label. Tumors were then washed, fixed, and analyzed for the presence and location of tumor cells, CEA expression, fluorescence, and radioactivity. Twenty-six of 29 tumor samples obtained from 10 patients contained malignant cells. Overall, fluorescence intensity was higher in tumor areas compared to adjacent non-tumor tissue parts (p < 0.001). The average fluorescence tumor-to-background ratio was 11.8 ± 9.1:1. A similar ratio was found in the autoradiographic analyses. Incubation with a non-specific control antibody confirmed that tumor targeting of our tracer was CEA-specific. Our results demonstrate the feasibility of this tracer for multimodal image-guided surgery. Furthermore, this ex vivo incubation method may help to bridge the gap between preclinical research and clinical application of new agents for radioactive, near infrared fluorescence or multimodal imaging studies.

Keywords: antibody; colorectal cancer; ex vivo; image-guided surgery; targeted imaging near infrared fluorescence; translational medicine.

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Conflict of interest statement

David M. Goldenberg is retired chairman and founder of Immunomedics, Inc., and IBC Pharmaceuticals, Inc., and holds royalty-bearing patents. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mean fluorescence intensity (arbitrary units) per pixel for tumor (green dots) and normal tissue (black diamonds) in individual tumors. Each green circle represents an included tumor. Vertical dashed lines separate patients. Note the higher fluorescence signal in all tumors compared to surrounding normal tissue (p < 0.001). The control condition (incubation with the non-specific antibody-conjugate DOTA-hIgG-IRDye800CW) shows no significant difference between tumor and normal tissue tracer accumulation (red circles and black open diamond; last two patients).
Figure 2
Figure 2
Example of an ROI for tumor (orange line) and surrounding tissue (pink line) as drawn on the H&E stained slide (A). (B) Consecutive slide with immunohistochemical CEA staining. (C) fluorescence flatbed image of the same slide as (A). (D) autoradiography image of the same slide as (A).
Figure 3
Figure 3
Method of ex vivo incubation of surgical tissue samples with 111In-DOTA-hMN-14-IRDye800CW. (A) surgical resection of tumor specimen and tissue preparation. (B) Overnight incubation with 111In-DOTA-hMN-14-IRDye800CW at 37 °C on orbital shaker with antibiotic additives. (C) Four hours washing of unbound antibody-conjugate at 4 °C. (D) Formalin-fixation, paraffin embedding and mounting of serial slices on glass slides for (immuno)histochemical analyses.
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