Development of a stereological method to measure levels of fluoropyrimidine metabolizing enzymes in tumor sections using laser scanning cytometry.
BACKGROUND
The enzymes thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) influence the activities of fluoropyrimidine anticancer drugs. The sensitivity of cancer cells to capecitabine, which is an oral, tumor-selective pre-prodrug of 5-fluorouracil may correlate better to the TP/DPD ratio than to levels of either enzyme alone. Our goal was to develop a quantitative immunofluorescent method for estimating the levels of TP, DPD, and their ratio in archival tumor sections.


METHODS
Mouse anti-TP and rat anti-DPD monoclonal antibodies were used for parallel indirect immunofluorescent staining. The fluorescence was measured using a laser scanning cytometer (LSC; CompuCyte, Cambridge, MA) in single cells and in sections prepared from cell lines and a human tumor. The phantom contouring feature of the LSC provided a stereologic approach for collecting the fluorescence intensity data from sections.


RESULTS
The relative fluorescence intensities measured in single cells or in sections of the cell lines, using single or double labeling, were similar, supporting the suitability of phantom contouring and two-color staining. Sections of the T-24 and ZR-75-1 cell lines placed on the same slide as the tumor section were used as internal standards for fluorescence measurements. The TP/DPD ratios measured in three cell lines correlated well with the cytotoxicity of 5'-deoxy-5-fluorouridine measured in vitro, indicating that the measurements are related to the biological activity of the drug.


CONCLUSIONS
Plotting the data as contour maps of the topologic distribution of fluorescence intensities in tumor sections allows subsequent histopathologic examination, which may reveal features of the tumors leading to high or low ratios of these enzymes. In addition, this method can be used for any drug target/metabolic system where the key components are known and suitable antibodies are available.
Megyeri
Attila
A
Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48202, USA.
Bacsó
Zsolt
Z
Shields
Anthony
A
Eliason
James F
JF
D000911:Antibodies, Monoclonal / Q000737:chemistry; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000032:analysis* / Q000378:metabolism; D004938:Esophageal Neoplasms / Q000201:enzymology / Q000473:pathology; D005467:Floxuridine / Q000494:pharmacology; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D047569:Laser Scanning Cytometry / Q000379:methods*; D009369:Neoplasms / Q000201:enzymology* / Q000473:pathology; D011743:Pyrimidines / Q000378:metabolism*; D013939:Thymidine Phosphorylase / Q000032:analysis* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000964:Antimetabolites, Antineoplastic; D005456:Fluorescent Dyes; D011743:Pyrimidines; D005467:Floxuridine; C029269:5-fluoropyrimidine; D042943:Dihydrouracil Dehydrogenase (NADP); D013939:Thymidine Phosphorylase; C025034:doxifluridine