Matrix‐Metalloproteinases in Head and Neck Carcinoma–Cancer Genome Atlas Analysis and Fluorescence Imaging in Mice

Abstract

Objective (1) Obtain matrix-metalloproteinase (MMP) expression profiles for head and neck squamous cell carcinoma (HNSCC) specimens from the Cancer Genomic Atlas (TCGA). (2) Demonstrate HNSCC imaging using MMP-cleavable, fluorescently labeled ratiometric activatable cell-penetrating peptide (RACPP). Study Design Retrospective human cohort study; prospective animal study. Setting Translational research laboratory. Subjects and Methods Patient clinical data and mRNA expression levels of MMP genes were downloaded from TCGA data portal. RACPP provides complementary ratiometric fluorescent contrast (increased Cy5 and decreased Cy7 intensities) when cleaved by MMP2/9. HNSCC–tumor bearing mice were imaged in vivo after RACPP injection. Histology was evaluated by a pathologist blinded to experimental conditions. Zymography confirmed MMP-2/9 activity in xenografts. RACPP was applied to homogenized human HNSCC specimens, and ratiometric fluorescent signal was measured on a microplate reader for ex vivo analysis. Results Expression of multiple MMPs including MMP2/9 is greater in patient HNSCC tumors than matched control tissue. In patients with human papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14 expression correlates with worse 5-year survival. Orthotopic tongue HNSCC xenografts showed excellent ratiometric fluorescent labeling with MMP2/9-cleavable RACPP (sensitivity = 95.4%, specificity = 95.0%). Fluorescence ratios were greater in areas of higher tumor burden (P <.03), which is useful for intraoperative margin assessment. Ex vivo, human HNSCC specimens showed greater cleavage of RACPP when compared to control tissue (P = .009). Conclusions Human HNSCC tumors show increased mRNA expression of multiple MMPs including MMP2/9. We used RACPP, a ratiometric fluorescence assay of MMP2/9 activity, to show improved occult tumor identification and margin clearance. Ex vivo assays using RACPP in biopsy specimens may identify patients who will benefit from intraoperative RACPP use.

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling

“…In addition, MMP14 also plays a key role in potentiating cancer cell migration [32, 33]. These results are in agreement with recent findings, which indicate a poor 5-year survival rate in patients with increased MMP14 expression regardless of HPV status and make it a potential target since its selective inhibition blocked tumour growth, invasion and angiogenesis [34, 35]. …”

FRET-trackable biodegradable HPMA copolymer-epirubicin conjugates for ovarian carcinoma therapy

“…The release of model drug Cy3 will result in decrease of FRET intensity that can be investigated via cell lysis using fluorescence spectrometry. According to the previous reports [44–46], the ratio, I Cy3 /I FRET , was calculated to quantify the FRET change, where I Cy3 and I FRET are the fluorescence intensity at 562 nm and 664 nm, respectively (excitation 520 nm). For the conjugate P-Cy3-Cy5, the ratio in medium alone was 2.35, and in initial stock was 1.97.…”

Fluorescence Identification of Head and Neck Squamous Cell Carcinoma and High-Risk Oral Dysplasia With BLZ-100, a Chlorotoxin-Indocyanine Green Conjugate

“…However, the currently available tools to identify dysplasia, such as toluidine blue and endogenous tissue fluorescence detection, demonstrate high false-positivity and the inability to distinguish high-risk from low-risk lesions [29–33]. Intravenous fluorescent-labeled targeting agents, based on protoporphyrin precursors [34], antibodies [35] and enzyme-activatable peptides [36], represent a promising strategy to deliver accurate, high-contrast identification of tumor with minimal side effects. However, there is limited study of these agents within the context of dysplasia [37–39]; only one other agent besides our present study has been reported in a model of oral dysplasia [40].…”