Matrix-metalloproteinases in head and neck carcinoma-cancer genome atlas analysis and fluorescence imaging in mice.

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.

Ratiometric activatable cell-penetrating peptides provide rapid in vivo readout of thrombin activation.

In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.

Real-time in vivo molecular detection of primary tumors and metastases with ratiometric activatable cell-penetrating peptides.

Management of metastatic disease is integral to cancer treatment. Evaluation of metastases often requires surgical removal of all anatomically susceptible lymph nodes for ex vivo pathologic examination. We report a family of novel ratiometric activatable cell-penetrating peptides, which contain Cy5 as far red fluorescent donor and Cy7 as near-infrared fluorescent acceptor. Cy5 is quenched in favor of Cy7 re-emission until the intervening linker is cut by tumor-associated matrix metalloproteinases-2 and 9 (MMP2,9) or elastases. Such cleavage increases the Cy5:Cy7 emission ratio 40-fold and triggers tissue retention of the Cy5-containing fragment. This ratiometric increase provides an accelerated and quantifiable metric to identify primary tumors and metastases to liver and lymph nodes with increased sensitivity and specificity. This technique represents a significant advance over existing nonratiometric protease sensors and sentinel lymph node detection methods, which give no information about cancer invasion.

Disturbed distribution of proliferative brain cells during lupus-like disease.

Brain atrophy and neuronal degeneration of unknown etiology are frequent and severe concomitants of the systemic autoimmune disease lupus erythematosus (SLE). Using the murine MRL/lpr model, we examined populations of proliferative brain cells during the development of SLE-like disease and brain atrophy. The disease onset was associated with reduced expression of Ki67 and BrdU proliferation markers in the dorsal part of the rostral migratory stream, enhanced Fluoro Jade C staining in the subgranular zone of the dentate gyrus, and paradoxical increase in density of Ki67(+)/BrdU(-) cells in the paraventricular nucleus. Protuberances containing clusters of BrdU(+) cells were frequent along the lateral ventricles and in some cases were bridging ventricular walls. Cells infiltrating the choroid plexus were Ki67(+)/BrdU(+), suggesting proliferative leukocytosis in this cerebrospinal fluid-producing organ. The above results further support the hypothesis that systemic autoimmune disease induces complex CNS pathology, including impaired neurogenesis in the hippocampus. Moreover, changes in the paraventricular nucleus implicate a metabolic dysfunction in the hypothalamus-pituitary-adrenal axis, which may account for altered hormonal status and psychiatric manifestations in SLE.

Lingual fat at autopsy.

OBJECTIVES/HYPOTHESIS: Obesity as measured by body mass index (BMI) has been shown to correlate with incidence and severity of sleep disordered breathing (SDB), but the actual mechanism underlying this relationship has not been defined. Pharyngeal obstruction from posterior displacement of a large, fat laden tongue is one mechanism that may explain this link. The objective of this study is to characterize the fat content within the tongue and then to determine whether tongue weight and percent of fat correlate with BMI and other metrics of obesity. STUDY DESIGN: This is a cross-sectional anatomic study performed at autopsy in 121 consecutive medical examiner cases. METHODS: Tongues were harvested, weighed, and sectioned. A standardized photograph was taken of each tongue in the midsagittal plane. The image was imported into ImageJ (NIH) and then digitally analyzed to estimate fat distribution and percent within the tongue. The measurements were divided into age and sex subsets and then examined for correlation with height, weight, BMI, organ weight, and abdominal subcutaneous fat thickness. RESULTS: This study includes data from 88 males, 27 females, and 6 children. The average tongue weight for males was 99 g, range 71 to 143 g; for females, 79 g, range 51 to 135 g; and for children, (mean age 4 yr) 38 g, range 15 to 81 g. Tongue weight correlated with BMI (r = 0.6, P < .0001). Percent of fat in the posterior tongue averaged 30 +/- 12% and correlated with BMI (r = 0.5, P < .0001) for both men and women. Percent of fat in the anterior tongue averaged 10 +/- 5% and correlated with BMI for males (P < .001). Anterior tongue fat percent did not correlate with BMI in females. CONCLUSION: Increase in tongue weight and percentage of fat, and therefore tongue volume, may explain why patients with weight gain have higher rates of SDB. Tongue weight, fat, and volume may also correlate with and explain Mallampati grades.