Morphometric analysis of bromodeoxyuridine distribution and cell density in the rat Dunning prostate tumor R3327-AT1 following treatment with radiation and/or hyperthermia

To monitor cellular response to single doses of radiation (RT) and/or local tumor hyperthermia (LTH) proliferation kinetics were determined in the anaplastic prostate adenocarcinoma R3327-AT1 grown in Copenhagen rats. Tumor-bearing animals were injected i.v. with a bolus of bromodeoxyuridine (BrdUrd), and at defined times after treatment the tumors were surgically removed, fixed and embedded in paraffin. BrdUrd incorporated into the DNA of S-Phase nuclei was detected on 4-6 mu m-thick tissue sections using a monoclonal anti-BrdUrd antibody followed by streptavidin-biotin and alkaline phosphatase as a reporter system. Cell nuclei were stained with the fluorescence dye DAPI (Diaminophenylindole). Morphometric analysis was performed using a computer-assisted Leitz-TAS/plus system. Depending on tumor size, up to 18,000 nuclei were routinely analyzed. Untreated tumors of standardized size (8-10 mm) exhibited a BrdUrd-labeling index (LI) of (6.9+/-1.6)%. In general, the LI was higher in the periphery than in the center, being more pronounced in larger tumors. After 6 Gy gamma-rays, the mean LI decreased to 1.8% (24 h) and rose afterwards to 5.4% by 168 h. Following LTH (43.5 degrees C, 35 min water bath), the mean LI rapidly decreased to 2% (8 h), rose to 9.8% (48 h), and plateaued at 6% after 168 h. A combined treatment consisting of irradiation (6 Gy) followed by LTH yielded smallest LI (2.4+/-0.18%) and lowest cell density (111+/-0.6 nuclei per field) by 168 h. The morphometric procedure was reliable and reproducible and can be used to characterize and compare the effects of different therapies on cell kinetics. Of particular value is that these analyses are done on an intact tissue architecture and hence enable a better interpretation of flow cytometric results of treatment-induced alterations within different topohistological regions in solid tumors. Moreover, the technique provides the basis for 3D reconstruction of the cellular activity and heterogeneity of experimental neoplasms.