Improved ex vivo fluorescence imaging of human head and neck cancer using the peptide tracer TPP-IRDye800 targeting membrane-bound Hsp70 on tumor cells.

Background: The primary goal of surgery in HNSCC is the complete resection of tumor cells with maximum preservation of normal tissue. The membrane Hsp70-targeting fluorescence labelled peptide TPP-IRDye800 represents a promising tool for real-time intraoperative tumor visualization, enabling the detection of true tumor margins, critical isles of high-grade dysplasia and LN metastases.
Methods: Membrane Hsp70 (mHsp70) expression on HNSCC cell lines and primary HNSCC was determined by flow cytometry and fluorescence microscopy using FITC-conjugated mAb cmHsp70.1 and TPP. TPP-IRDye800 was sprayed on freshly resected tumor material of immunohistochemically confirmed HNSCC and LN metastases for tumor imaging. TBRs were compared using TPP-IRDye800 and Cetuximab-IRDye680, recognizing EGFR.
Results: mHsp70 expressing HNSCC cells specifically bind and internalize TPP in vitro. The TBR (2.56 ± 0.39) and AUC [0.98 CI, 0.95-1.00 vs. 0.91 CI, 0.85-0.97] of TPP-IRDye800 on primary HNSCC was significantly higher than Cetuximab-IRDye680 (1.61 ± 0.39) (p = 0.0068) and TPP-IRDye800 provided a superior tumor delineation. Fluorescence imaging showed higher AUC values than a visual inspection by surgeons [0.97 CI, 0.94-1.00 vs. 0.92 CI, 0.88-0.97] (p = 0.048). LN metastases could be visualized using TPP-IRDye800. Real-time tissue delineation was confirmed using the clinically applied KARL-STORZ imaging system.
Conclusion: TPP-IRDye800 is a promising fluorescence imaging probe for HNSCC.
Competing Interests: Competing interests: GM has ownership interest as a patent inventor (PCT/EP2015/063697) and is CSO of multimmune GmbH, Munich, Germany providing the cmHsp70.1 mAb. Ethics approval and consent to participate: All patients gave their written consent to image their excised tissue and to provide blood and tumor biopsies. The study protocol was approved by the Ethics Committee of the Klinikum rechts der Isar (MRI) of the Technical University of Munich (2021-132). All analyses were conducted in accordance with the Declaration of Helsinki.
(© 2024. The Author(s).)