Potential prognostic indicators for the radiotherapy of cervical carcinoma

Biopsies from 117 patients with proven cervical carcinoma were taken under anaesthesia immediately prior to radiotherapy. The Courtenay Mills soft agar clonogenic assay was used to determine the colony forming efficiency (CFE) of tumour cells from disaggregated tumours and the survival of these cells following radiation. Validation of the assay was carried out by demonstrating linearity of colony formation and production of radiation survival curves. The mean CFE was 0.18±0.49% (±lsd) with a range of 0.003 - 4.28% (based on total viable nucleated cell counts) for 84 (72%) specimens. No significant association was demonstrated between in vitro growth and either clinical stage (r=0.02) or tumour differentiation (r=0.08). A wide range of values (0.13-0.97) for surviving fraction at 2Gy (SF2) was obtained with a mean value 0.44 (sd=0.19) for 77 tumours. There were statistically significant differences between the individual tumours (p=<. 001). Heterogeneity in intrinsic radiosensitivity was not demonstrated (p=0.3) when multiple biopsies were processed independently from 18 tumours. From analysis of variance of the SF2 results it appears that the surviving fraction below 0.4 and those above 0.7 which show significant differences in radiosensitivity between pairs of tumours (p=OO5). Differential cell counts were made on cytospin preparations of tumour cell suspensions. There was no correlation between either CFE or SF2 (r = -0.05, r=0.15, respectively) and the degree of lymphocyte (r = 0.12) or macrophage (r = 0.001) infiltration. Ki67 staining of 29 specimens gave a mean proportion of positively stained nuclei of 20.5% (sd=23). The Ki67 index was not correlated with tumour stage (r=0.58), differentiation (r=0.02), CFE (r=0.04) or SF2 (r="0.07). Vascularity was assessed on paraffin sections of 87 tumours. The mean intercapillary distance (ICD) was 240μm (sd=37pm) and mean proportion of vessels was 2.68% (sd=1.64%). Clinical data from 35 patients with 2 year minimum follow-up revealed no significant difference between the mean ICD or mean proportion of vessels for the group of patients which had died or recurred and the group which remained disease free using t-tests (t=0.74, p=0.47, DF=32; t= 0.6, p=0.55, DF=29 respectively)

Characterisation and in vitro drug sensitivities of human transitional cell carcinoma cell lines: evaluation of their use as a model system for bladder cancer

The concept that continuous cell lines derived from one histological type of tumour provide an in vitro model system for that disease was examined. Biological characteristics and in vitro drug sensitivities of lines derived from transitional cell carcinomas (TCC) of the human bladder are described. The isozyme phenotype, cytological appearance, cell kinetics, colony-forming efficiency, tumorigenicity and monoclonal antibody reactivities of twenty-two urothelial lines were assessed. Three types were identified; 1) fourteen distinct lines derived from different patients, 2) five cross-contaminated with one of the distinct lines (T24) and 3) three non-tumorigenic lines. Xenograft morphology and isozyme pattern were used to select representative TCC cell lines. Ten clonogenic assay procedures for measuring cytotoxicity were compared using one cell line (RT112) exposed to adriamycin and methotrexate. Significant differences between dose-response curves were obtained for the same drug indicating methods should be standardised. The relative cytotoxicities of a 24h exposure to twelve chemotherapeutic drugs against RT112 cells were compared using a clonogenic assay. Drug concentrations reducing clonogenic cell survival by 10% ranged from 2.9 ng/ml for mitoxantrone to 27.0 pg/ml for hydroxyanisole. The range and reproducibility of in vitro sensitivities to adriamycin and methotrexate were compared using eight distinct lines and four lines cross-contaminated with T24. The cross-contaminated lines had similar sensitivities, demonstrating stability during long-term culture. However there was a broads pectrum of sensitivities (>50 fold) amongst the distinct lines to methotrexate and a narrow range (<3 fold) to adriamycin. In summary, these data show that cell lines derived from human bladder tumours can retain certain biological characteristics of the tumours of origin. Taken in conjunction with published data, these results indicate that cell lines also retain the pattern of drug sensitivities of the tissue of origin, and if the differences in pharmacokinetics in vivo can be taken into account, might be used for screening new agents

Anchorage-independent growth of human tumour cells

The cell culture clonogenic assay appears to have considerable application in predicting individual cancer patient response to chemotherapy. There are several obstacles in the way of its successful application, however, including problems of extrapolating in vitro tests to the in vivo situation, and technical problems related to successful selective in vitro growth of primary human tumour cells. The work in this thesis addresses this latter area, using a human carcinoma line RPMI 2650 as a model system. Different disaggregation methods were compared; after short-term 0 incubation at 37 C, trypsin (0.25%) - EDTA (0.02%) produced the best viable single - cell suspension of RPMI 2650 cells. Following dissociation, RPMI 2650 cells and normal human fibroblasts were found to reaggregate spontaneously to form clusters. Clumps and clusters in cell suspensions contributed to final colony forming efficiencies; the significance of clumps and clusters in the clonogenic assay is discussed. Use of low gelling temperature agaroses improved colony forming efficiencies under some conditions examined. Use of ultrapure water in the preparation of semi-solid media also improved colony formation. Different incubation atmospheres were compared; RPMI 2650 colony formation was improved when the oxygen concentration was reduced from 20% to 3%. Colonies did not, however, grow under anaerobic conditions. Medium without foetal calf serum did not support RPMI 2650 colony formation; growth was also poor in 1% and 2% f.c.s. Inclusion of HITES supplements did not reduce the requirement for serum. Other growth factors tested include TCGF, EGF, insulin and hydrocortisone. Use of feeder cells improved colony forming efficiencies considerably; best results were obtained with mitomycin C-treated feeder cells. Inhibition was observed where high densities of feeder cells were used with high test cell densities. The contribution of cell cycle to final colony formation was examined but did not appear to be a contributory factor to the low cloning efficiencies observed. Normal human fibroblasts did not grow in either agar, agarose or methycelluloses, thus confirming the selective nature of the assay used in this laboratory. Colony formation in agarose was inhibited at a lower ouabain concentration than in monolayer; thioguanine inhibited colony formation in agarose and monolayer at the same toxic level. The significance of these findings is discussed and makes some contribution toward an understanding of the nature of anchorage-independent growth