| Background: High risk human papillomavirus (HPV) infections are considered a main contributor to the development of epithelial carcinoma, because HPV viral sequences are detected in more than 95% of all cervical and 26% of oropharyngeal carcinomas. Among the different proteins encoded by the HPV viral genome, HPV E6 and E7 have the functional ability to deregulate the cell cycle and to induce malignant transformation by affecting the p53 and pRB pathways, respectively. Therefore, correlating E6 and E7 expression with specific morphological and metabolic changes that occur in epithelia at the cellular and tissue level may help identify new biomarkers useful in epithelial cancer diagnosis. Methods: In this study, employing a tissue engineering approach, we correlate pre-cancerous changes induced specifically by E6 and E7 with biochemical and morphological signatures that are optically detectable. Specifically, we constructed and characterized epithelial tissues consisting of either normal human foreskin epithelial keratinocytes (HFKs) or HFKs expressing HPV E6, E7 or E6/E7 oncoproteins. We collected high resolution two-photon excited fluorescence (TPEF) images at 755 and 860 nm excitation and at 455+/-35 nm and 525+/-25 nm emission, detecting fluorescence contributions mostly from keratin, oxidized lipids, reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD). The NADH and FAD signals were used to calculate the redox ratio ([FAD]/([NADH]+[FAD]), which provides a robust readout of cell metabolic activity. Results: We found that the redox ratio as a function of depth in tissues expressing E6, E7 and E6E7 exhibits a relative shift towards higher values ranging from 0.61 to 0.68+/-0.04 for E6, from 0.65 to 0.7+/-0.035 for E6E7 and from 0.7 to 0.73+/-0.025 for E7 expressing tissues compared to those found in normal tissues, which range from 0.55 to 0.68+/-0.035. Our redox values suggest that E6 and E7 induce metabolic stresses, whose cumulative effect is a decrease in NADH concentration relative to that of FAD. Furthermore, our data reveal that the effects of E6 and E7 on metabolism are not additive and that E7 causes the major increase in redox ratio. Conclusion: The results show the potential of TPEF imaging to serve as a tool for assessing biochemical changes that occur at the very early stages of cancer development. Such information could be useful not only to improve diagnosis clinically, but also to understand the origins of the detected optical signals and the relationships between changes in cell structure and function. |
