Early detection of squamous cell carcinoma in carcinogen induced oral cancer rodent model by ratiometric activatable cell penetrating peptides.

OBJECTIVES: Ratiometric cell-penetrating-peptides (RACPP) are hairpin-shaped molecules that undergo cleavage by tumor-associated proteases resulting in measurable Cy5:Cy7 fluorescence ratiometric change to label cancer in vivo. We evaluated an MMP cleavable RACPP for use in the early detection of malignant lesions in a carcinogen-induced rodent tumor model. METHODS: Wild-type immune-competent mice were given 4-nitroquinoline-oxide (4NQO) for 16weeks. Oral cavities from live mice that had been intravenously administered MMP cleavable PLGC(Me)AG-RACPP were serially imaged from week 11 through week 21 using white-light reflectance and Cy5:Cy7 ratiometric fluorescence. RESULTS: In an initial study we found that at week 21 nearly all mice (13/14) had oral cavity lesions, of which 90% were high-grade dysplasia or invasive carcinoma. These high-grade lesions were identifiable with white light reflectance and RACPP Cy5:Cy7 ratiometric fluorescence with similar detectability, Area Under Curve (AUC) for RACPP detection was 0.97 (95% Confidence interval (CI)=0.92-1.02, p<0.001), sensitivity=89%, specificity=100%. In a follow up study, oral cavity lesions generated by 4NQO were imaged and histologically analyzed at weeks 16, 18 and 21. In this study we showed that RACPP-fluorescence detection positively identified 15 squamous cell carcinomas (in 6 separate mice) that were poorly visible or undetectable by white light reflectance. CONCLUSIONS: RACPP ratiometric fluorescence can be used to accurately detect carcinogen-induced carcinoma in immunocompetent mice that are poorly visible or undetectable by white light reflectance.

Combined TP53 mutation/3p loss correlates with decreased radiosensitivity and increased matrix-metalloproteinase activity in head and neck carcinoma.

OBJECTIVE: Patients with head and neck squamous cell carcinoma (HNSCC) containing TP53 mutation and 3p deletion ("double-hit") have poorer prognosis compared to patients with either event alone ("single-hit"). The etiology for worse clinical outcomes in patients with "double-hit" cancers is unclear. We compared radiosensitivity of cell lines containing both TP53 mutations and deletion of Fragile Histidine Triad (FHIT, the gene most commonly associated with 3p deletion) to "single-hit" lines with only TP53 mutation. We compared radiosensitivity in a "single-hit" cell line with TP53 mutation converted to "double-hit" using RNA interference targeting FHIT. Finally, we compared matrixmetalloproteinase-2/9 (MMP-2/9) activity, a previously-established biomarker for tumor aggressiveness, in xenograft tumors derived from these cell lines. MATERIALS/METHODS: TP53 mutation and FHIT deletion profiles of HNSCC lines were established using Cancer Cell Line Encyclopedia (CCLE). We used RNA-interference to convert a "single-hit" cell line (SCC4) to "double-hit". Cultured cells were examined for radiosensitivity and cisplatin sensitivity. MMP-2/9 activity was evaluated in "double-hit" versus "single-hit" tumors using ratiometric activatable cell-penetrating peptide (RACPP) in tongue (n=17) and flank xenografts (n=4). RESULTS: Radiotherapy caused greater double-stranded DNA breaks in "single-hit" vs naturally occurring and engineered "double-hit" cells. In-vivo, "double-hit" xenografts demonstrated higher MMP-2/9 activity compared to "single-hit" xenografts (p<0.01). There was no difference in cisplatin sensitivity between the cell lines. CONCLUSIONS: TP53 mutation combined with FHIT deletion correlates with decreased radiosensitivity in HNC cell lines. Xenograft from "double-hit" cells exhibit increased MMP-2/9 activity. These findings may in part account for the worse clinical outcome seen in patients with HNSCC "double-hit" tumors.