Bioluminescence imaging has evolved as a powerful tool for monitoring biological processes in vivo. As transmission efficiency of light through tissue increases greatly for wavelengths above 600 nm we examined whether a redshifted codon-optimized firefly luciferase (lambdamax=615 nm) could be successfully employed as a sensitive reporter in mammalian cells. To this end, unmodified codon-optimized luciferase (lambdamax=557 nm) as well as the red-emitting S284T mutant luciferase were expressed simultaneously in human glioma cells in vitro as well as in quadriceps muscles of mice in vivo. We show here that activity of the redshifted enzyme in human glioma cell culture approached approximately one-fourth of that seen with the unmodified enzyme. In contrast, light emission by the red-emitting luciferase in vivo was generally more efficient than that produced by its unmodified counterpart, most likely due to reduced absorption of red light by tissue. The mean ratio of light emission produced by the redshifted luciferase to that of the unmodified enzyme in vivo was approximately 3. Application of this new redshifted luciferase together with other optical reporters may be of considerable importance to biological research as it allows for imaging of deeper tissues as well as simultaneous monitoring of two molecular events in vitro and in vivo if appropriate filter sets are employed.

Background: Positron emission tomography (PET) offers an opportunity to examine noninvasively cellular functions with different tracers. [¹⁸F]Fluorodeoxyglucose (FDG) is most commonly used in identifying malignant tumors. Several tumor biologic characteristics (tumor cell viability, growth faction, treatment response to radiation, cell membrane dysfunction, recurrence rate) are suggested to be characterized by [¹⁸F]FDG PET. The aim of this study was to assess which other tumor biologic characteristics of squamous-cell carcinoma of the head and neck are correlated with [¹⁸F]FDG PET. Methods: [¹⁸F]FDG PET was performed in 14 patients with squamous-cell carcinomas of the upper digestive tract (TNM classification T₂–T₄, N₁–N₃). After attenuation correction, predefined areas of the tumor were semiquantitatively analyzed by the technique of the region of interest and calculated as standard uptake values (SUV). Afterwards, 5 biopsies of different tumor regions were obtained during endoscopy in each patient under general anesthesia, and a correlation between SUV of [¹⁸F]FDG PET and tumor biologic parameters was attempted. These parameters included: quantitative DNA measurements (i.e. 2c deviation index, 5c exceeding rate), immunohistochemical assessment of growth fraction (i.e. Ki67-MIB-1, PCNA) along with morphological tumor front grading. Results: The results revealed a marked variation of proliferation and cellular differentiation in various regions of the tumor for all parameters examined. There was a close correlation between [¹⁸F]FDG uptake and growth fraction (r = 0.83 for Ki67-MIB-1 and r = 0.8 for PCNA). A poor correlation was found between DNA aneuploidy (r = 0.4) or tumor front grading (r = 0.12) and [¹⁸F]FDG uptake. Conclusions: Our results confirm previous clinical and histologic observations that squamous-cell carcinomas of the upper digestive tract are heterogeneous tumors. Ki67 antigen, which has been shown to be of predictive value for proliferation and individual prognosis, correlated with [¹⁸F]FDG uptake. Using [¹⁸F]FDG PET, the main proliferation centers of inhomogeneous squamous-cell carcinomas could be identified with possible clinical implications for patient management.

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Specimens obtained from five different tumor regions in 12 patients who underwent surgery for squamous cell carcinoma (SCC) of the oropharynx were examined. The evaluation of each biopsy included quantitative DNA measurements based on image analysis, immunohistochemical assessment of proliferations markers (i.e., Ki67-MIB1, proliferating cell nuclear antigen [PCNA]), and morphological tumor-front grading. From single cell measurements, several DNA indices were derived which are known to reflect tumor aneuploidy. The results revealed a marked variation of proliferation and cellular differentiation in different regions of tumors and a wide intraindividual variation between particular tumors for all markers examined. There was good correlation between DNA data and proliferative cell fractions (Ki67 score, PCNA score). With the use of diagrams, three-dimensional distribution of proliferation rates and markers reflecting tumor aggressiveness within each tumor was obtained. The results confirmed previous clinical and histological observations that SCCs of the oropharynx are heterogeneous tumors. One might expect that the regions with increased proliferation and aggressiveness may predict the location of possible tumor recurrence.

Although several cytogenetic events of the tumor progression cascade have been identified in the past, the specific types of chromosomal alterations that lead to the development of lymph node metastases are still unknown. Operative specimens of 20 patients (10 patients with metastasizing tumors, 10 patients with nonmetastasizing tumors) with squamous cell carcinomas of the oropharynx and hypopharynx, along with the corresponding lymph node metastases, were investigated by quantitative DNA measurements and comparative genomic hybridization (CGH). Nonmetastasizing tumors (N0) displayed overrepresentations on chromosomes 10q (8 cases); 5p (7 cases); 3q and 20q (6 cases each); 8q (5 cases); 1p and 21q (4 cases each); 7p and 20p (3 cases each); and 2p, 15q, and 19q (2 cases each). Loss of chromosomal material was found on 5q, 9p, and 14q (2 cases each). Metastasizing tumors (N+) demonstrated overrepresentations on chromosomes 5p, 15q, and 22q (6 cases each); 3q and 11q13 (5 cases each); 20p and 21q (4 cases each); and 10q (3 cases). In 2 cases, an overrepresentation of the chromosomal arm 3q was accompanied by a loss of chromosomal arm 3p. Less frequent overrepresentations were observed on chromosomes 1q and 17q. Deletions were found on chromosomes 18q (3 cases), 3p, 4q, 5q, and 19p (2 cases each); and sporadic deletions occurred on 2q, 6q, 8p, 9p, 10p, 13q, 14q, 15q, and 16q. Whereas overrepresentations on chromosomes 1p and 7p occurred exclusively in N0 tumors, overrepresentations on chromosomes 1q, 11q, and 22q, along with deletions on 18q, were only observed in N+ tumors. Quantitative DNA measurements revealed a significantly higher percentage of aneuploid cells and a higher degree of DNA entropy in the N+ tumors. Chromosomal overrepresentations on chromosomes 1q, 8q, 11q, 18q, and 19q occurred more frequently in the metastases than in the corresponding primary tumors. Pairwise analysis of chromosomal alterations in the primary tumors and associated lymph node metastases revealed a genetic relationship, although a greater number of chromosomes on average were affected in the lymph node metastases. Quantitative DNA measurements demonstrated greater aneuploid values in the metastases. Recurring patterns of chromosomal alterations in N0 and N+ tumors were demonstrated in this study. In general, metastasizing tumors are characterized by overrepresentations on chromosomes 11q13 and 22q, and deletions on 18q. These aberrations suggest an elevation along the tumor progression cascade.