CELL KINETIC-ANALYSIS OF MURINE SQUAMOUS-CELL CARCINOMAS - A COMPARISON OF SINGLE VERSUS DOUBLE-LABELING USING FLOW-CYTOMETRY AND IMMUNOHISTOCHEMISTRY

The study was originally set up to measure accurate cell kinetic param eters in two murine squamous cell carcinomas (scc) for comparison with radiobiological data on proliferation during radiotherapy. The tumour s, AT84 and AT478, were both moderately well differentiated aneuploid scc. In the course of the study, several comparisons of techniques wer e made in two different centres. This paper reports on the results of those comparisons involving two different detection methods (flow cyto metry and immunohistochemistry), single vs double labelling, and in vi vo and in vitro labelling, the latter using tissue slices incubated un der high pressure oxygen. Pulse labelling studies with bromodeoxyuridi ne (BrdUrd) showed that the labelling indices (LI) were not significan tly different after in vitro or in vivo labelling. In addition, the fl ow cytometry (FCM) and immunohistochemistry (IHC) methods also gave la belling indices which were not significantly different. Only tumour ce lls were analysed in these studies by selecting cells on the basis of aneuploidy (FCM) or morphology (IHC). The DNA synthesis time of the tu mour cells were analysed by both techniques. For FCM, the Relative Mov ement method was used (Begg et al., 1985). For IHC. a double labelling method was used, employing BrdUrd and triated thymidine (H-3-TdR) adm inistered several hours apart, detected simultaneously using immunoper oxidase and autoradiography, respectively. When both labels were admin istered in vivo, there was good agreement for T(s) between the FCM and IHC methods. Attempts were also made to measure T(s) in vitro using b oth techniques. With double labelling, it was found that cells did not take up the second label. implying a failure of cycle progression. Th is was confirmed by FCM results, showing no movement of labelled cells through the S-phase, despite an initially high uptake. This could not be influenced by lowering the DNA precursor concentration or by addin g foetal calf serum. This indicates that DNA synthesis times are diffi cult or impossible to measure in vitro in fresh tumour explants. Final ly, the double labelling IHC method allowed intratumoural variations o f both LI and T(s) to be studied. Both parameters were found to vary m arkedly throughout the tumour volume, particularly for larger tumours (600 mg), giving calculated local potential doubling time values (T(po t)) ranging from 1-7 days.