Fluorescence Imaging of Head and Neck Squamous Cell Carcinoma

Abstract

Objectives:

(1) Demonstrate that head and neck squamous cell carcinoma (HNSCC) can be fluorescently imaged using a matrix metalloproteinase (MMP)-cleavable, ratiometric activatable cell-penetrating peptide (RACPP). (2) Correlate extent of tumor involvement with fluorescent signal.

Methods:

Increased expression of MMP-2 and MMP-9 has been well documented in multiple cancers. We developed an injectable probe (RACPP) that exhibits ratiometric fluorescence (increased Cy5:Cy7 ratio) when cleaved by these proteinases. To examine the utility of MMP2,9-cleavable RACPPs in HNSCC, mice were injected with 5 human HNSCC cell lines to establish orthotopic tongue xenografts (n = 22). Tumor-bearing mice were imaged in-vivo after intravenous RACPP injection. Fluorescent signal was correlated with histology by a blinded pathologist. Gelatinase zymography confirmed MMP-2,9 activity in these xenografts. For ex-vivo analysis of human HNSCC specimens, RACPP was applied to homogenized samples (n = 5), and fluorescence was measured on a microplate reader.

Results:

Orthotopic tongue HNSCC xenografts showed excellent ratiometric fluorescent labeling with MMP2,9-cleavable RACPP (sensitivity = 95.4%, specificity = 95.0%). Signal intensity, as defined by ratiometric contrast, was greater in areas of higher tumor burden (P < .03). Ex vivo human HNSCC specimens treated with MMP2,9-cleavable RACPP also had increased signal intensity when compared to normal human fat (P = .005).

Conclusions:

Fluorescent labeling with MMP2,9-cleavable RACPP is an effective way to visualize HNSCC in-vivo in a murine model, and signal intensity correlates with tumor burden. RACPPs have the potential to improve occult tumor identification and margin clearance in HNSCC. Ex-vivo assays using biopsy specimens may help identify patients who will benefit from intraoperative RACPP use and may be useful in retrospective analyses.