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. (Figure presented.)