Do cell lines in vitro reflect the properties of the tumours of origin? A study of lines derived from human melanoma xenografts.

The characteristics of 7 human melanoma cell lines were compared with those of the xenografts from which they were established. The ultrastructure, melanin content, isozyme pattern and chromosome numbers of the cell lines were closely similar to those of the corresponding xenografts. The different cell lines gave rise to colonies in soft agar of size and morphology similar to the parent xenografts, and the plating efficiencies were clearly correlated. However, no correlation was found between the growth rates in vivo and either the doubling times and saturation densities in monolayer cultures or the plating efficiencies in soft agar. Moreover, one of the cell lines lost its tumorigenic ability upon establishment in culture. Thus, although the properties of the cell lines by and large reflected those of the parent xenografts, important inconsistencies were seen. The data emphasize that extrapolations from continuous cell lines in vitro to tumour cells in vivo are not necessarily valid. A high content of cellular fibronectin was correlated with a compact colony morphology in soft agar and rapid attachment and spreading on plastic. The growth rates and cellular morphology of the cell lines were strongly influenced by TPA, DMSO, retinoic acid and theophylline, but not by alpha-melanocyte-stimulating hormone. A murine cell line established from one of the xenografts grew in soft agar and produced sarcoma in mice. The malignant murine cells had arisen by transformation of murine stromal cells during the first subcultures in vitro, possibly caused by a factor produced by the human melanoma cells

Predictive chemosensitivity testing in malignant melanoma: reliable methodology--ineffective drugs.

A retrospective and a prospective trial were carried out in patients with malignant melanomas to investigate the predictive value of an in vitro chemosensitivity assay based on the Courtenay and Mills soft agar cultivation method. Evaluable in vitro chemosensitivity data for the three agents DTIC, CCNU, and vinblastine were obtained in 153 cases. In the retrospective study in which the patients received chemotherapy without prior knowledge of the test results, 50 in vitro/in vivo correlations (40 patients) were made, and in the prospective study, where patients received the single agent most active in vitro, 55 correlations (45 patients) were performed. In both studies the sensitivity of the test (the ability to identify patients who will respond to chemotherapy) was approximately 100% and the specificity (the ability to identify patients who will not respond) was 87-98%. Depending on whether 'no change' and 'mixed response' were classified as sensitivity or resistance, the predictive value of a negative test was approximately 100% and that of a positive test 37.5-87.5%. The response rate was low in both series, and although it was somewhat higher in the prospective than in the retrospective trial, the difference was not significant. The median survival time was not significantly different in the two treatment series. We conclude that the chemosensitivity assay here used is reliable and has predictive value, but that the chemotherapeutic agents currently available for treatment of melanoma are too ineffective to warrant routine use of the assay in this disease

A human melanoma cell line established from xenograft in athymic mice.

A human melanoma cell line with unusually high growth potential was established from a xenograft growing in athymic mice. When xenograft fragments were cultured in vitro, melanoma cells grew out rapidly without any contamination of mouse stromal cells. An established cell line, FME, derived from this tumour, grew both in monolayer and in shaker suspension culture with doubling times of about 20 h. The cells grew easily at low serum concentrations and could even be cultured in serum-free medium supplemented with insulin and transferrin. The cultured cells were hyperdiploid, as were the cells of the xenograft. The cells grew easily in soft agar and formed tumours in athymic mice. When growing exponentially, the cells were almost unpigmented, but when grown to high density, their melanin content increased. Upon treatment with dimethyl sulphoxide (DMSO), retinoic acid and theophylline, as well as with the tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), the cells showed growth inhibition and increased melanin synthesis

Chemosensitivity measurements of human tumour cells by soft agar assays are influenced by the culture conditions.

To investigate the influence of culture conditions on the in vitro responses of tumour cells to anticancer drugs, the sensitivities observed with the soft agar methods of Hamburger & Salmon (1977) (H-S) and of Courtenay & Mills (1978) (C-M) were compared. In all cases the ID50 values were determined from dose-response curves. Six human tumour cell lines exposed to 10 different agents, and 9 patients' melanomas exposed to 5 different agents, were examined. In the studies of cell lines the H-S method gave higher sensitivity values than the C-M method in 38 out of 52 cases, whereas in 14 cases the results were the same. In the patients' tumours the H-S method gave higher sensitivity in 21 of 35 cases, equal sensitivity in 11, and lower sensitivity in 3 cases. In many instances the ID50 values obtained with the two test systems differed by factors of 10 or more, both in the case of cell lines and tumour specimens. Systematic alterations in the culture conditions indicated that the presence or absence of rat erythrocytes is the most important factor responsible for the differences observed. Also, other factors, such as supplements (in the H-S method) and the use of different serum types, appeared to influence both colony growth and chemosensitivity

Selection of tumour cell subpopulations occurs during cultivation of human tumours in soft agar. A DNA flow cytometric study.

To examine whether selection of tumour cell subpopulations occurs during cultivation in soft agar, we compared in 23 human tumours of different histological types the DNA content of cells from colonies formed in soft agar (method of Courtenay and Mills, 1978) with that of the original tumour cells. The ploidy as well as the fraction of cells in S phase were determined from DNA histograms after staining of the nuclei with a propidium-iodide procedure and flow cytometric recordings. In 8 of 17 aneuploid tumours analysed, specific aneuploid subpopulations disappeared during cultivation or new aneuploid populations, not demonstrable in the original cell suspensions, appeared in the colonies. In 9 cases identical aneuploid populations were found in the colonies and the tumours. In one of 6 diploid tumours examined, aneuploid cell populations not revealed in the original cell suspension, were found in addition to diploid cells, whereas 5 tumours gave rise to colonies containing a purely diploid population. The results show that in a variety of human malignant tumours cultivation in soft agar may select specific aneuploid tumour cell populations

Comparison of two soft-agar methods for assaying chemosensitivity of human tumours in vitro: malignant melanomas.

Two soft-agar methods for assaying chemosensitivity of human cancers in vitro were compared with respect to colony morphology, plating efficiency (PE) and chemosensitivity of human melanomas. In 9 xenografts and 9 patients' biopsy specimens Method A (essentially that of Courtenay & Mills, 1978) gave considerably higher PE that Method B (essentially that of Hamburger & Salmon, 1977) and, in contrast to Method B, the number of colonies was proportional to the number of cells plated. Evidence was obtained that the observed differences in PE could be attributed to the low O2 concentration and the presence of rat red blood cells in Method A. Colony morphology was similar in the 2 assays. When cells from 4 xenografted melanomas were treated in vitro with DTIC, CCNU, vinblastine and abrin, and the inhibition of colony formation was assayed concurrently in the 2 soft-agar methods, the tumour cells appeared to be more sensitive to 3 of the drugs in Method B than in A. The results demonstrate that chemosensitivity data obtained with the 2 assays cannot be directly compared

Colony forming ability of human breast carcinomas: lack of prognostic significance.

To study whether colony growth in vitro reflects the prognosis of breast cancer patients, specimens from a total number of 138 patients with primary breast carcinomas were cultivated in the Courtenay-Mills soft agar method. The plating efficiency (PE) values were related to various clinical and histopathological parameters. No significant correlation was found between colony forming ability and menopausal status, histopathology, TNM-status or steroid hormone receptor status. The crude survival of the patients was not significantly correlated to the in vitro growth of the tumours; neither was there any difference in relapse-free survival between patients whose tumours failed to grow in vitro and those having growing tumours (PE greater than 0). A multivariate survival analysis of 115 patients with primary tumours without distant metastases revealed that the PE was not a significant independent prognostic indicator, as it gave no additional prognostic information above that of node and ER status. It is concluded that routine measurement of colony formation in vitro is not warranted in the management of breast cancer

Microencapsulated octreotide pamoate in advanced gastrointestinal and pancreatic cancer: a phase I study.

Fourteen patients suffering from advanced colorectal (n = 7), pancreatic (n = 4) or gastric (n = 3) carcinomas received treatment with microencapsulated octreotide pamoate 90 mg i.m. every 4 weeks (n = 4), 160 mg i.m. every 4 weeks (n = 4) or 160 mg i.m. every 2 weeks (n = 6). Two patients had stable disease, one for 4 and one for 6 months. Plasma insulin-like growth factor (IGF)-I decreased by 49-53%, IGF-II by 27-37% and total IGF-binding protein (IGFBP)-3 by 16-19%, whereas IGFBP-1 increased by 35-55%. Insulin and C-peptide levels decreased by 29-38% and 41-46% respectively. A non-significant decrease in urinary GH secretion and an increase in the ratio of fragmented to intact IGFBP-3 as well as IGFBP-3 protease activity was seen. The increase in IGFBP-3 fragmentation correlated negatively with alterations in IGF-I and IGF-II (P < 0.05). We conclude that microencapsulated octreotide administered in doses up to 160 mg every 2 weeks is well tolerated and has pronounced effects on several components of the IGF system in plasma. In addition, changes in IGFBP-3 protease activity because of cancer may contribute to alterations in IGF-I and -II, indicating the importance of measuring this parameter in addition to IGFs and IGFBPs when evaluating alterations in IGF-I

Cultivation of human breast carcinoma in soft agar. Experience with 237 fresh tumour specimens.

A total of 237 breast carcinomas have been studied with the Courtenay-Mills (C-M) soft agar method. Cell yields and plating efficiencies (PE) were recorded after various enzyme treatments. The highest cell yields and PEs were obtained with the combination of collagenase 0.5%, hyaluronidase 1000 IE ml-1 and DNase 0.1% and an incubation time of 2 h. Eighty percent of the specimens gave greater than 10 colonies, and 60% formed greater than 30 colonies permitting chemosensitivity studies. The C-M method gave significantly higher PEs than the Hamburger-Salmon (H-S) method. Hormone supplements (insulin, oestradiol, progesterone, hydrocortisone) and also reduced agar concentrations (less than 0.3%) gave marginal stimulation of colony formation. In chemosensitivity studies involving doxorubicin, vincristine and 4-OOH-cyclophosphamide, the C-M method gave dose-response relationships without plateaus

Amplification and protein over-expression of the neu/HER-2/c-erbB-2 protooncogene in human breast carcinomas: relationship to loss of gene sequences on chromosome 17, family history and prognosis.

c-erbB-2 gene amplification and protein over-expression were investigated in 89 primary tumours and 24 metastases from Norwegian breast cancer patients. Amplification occurred in 22.5% of the primary tumours and 50% of the metastases. The amplification was negatively correlated to the oestrogen receptor (ER) content in both the primary tumours and the metastases. No significant differences between amplified and non-amplified tumours were observed with regard to node status, clinical stage, tumour size or menopausal status, although correlations of borderline significance were found between node status, clinical stage and high degree of gene amplification. All the amplified tumours were of the invasive ductal type. Follow-up data of patients observed for more than 1 year showed a significantly higher recurrence rate in the c-erbB-2 amplified group. Allele loss of chromosome 17p and of 7q was seen in 55% and 48% of the tumours respectively. No significant correlation was found between these losses and clinico-histological parameters. More than 50% of the tumours with a loss of 17q sequences had an amplification of c-erbB-2 which is located on 17q12-21, indicating that only one of the chromosomes may be involved in the amplification of the c-erbB-2. A trend towards a correlation between loss of 17q and high degree of amplification were found. No correlation was found between positive family history of breast cancer and c-erbB-2 gene amplification, nor loss of 17p or 17q sequences. Our data support the hypothesis that amplification correlates with aggressive tumour behaviour, and thus may be used as a prognostic factor in breast carcinomas. The allele losses on 17p and 17q points to tumour suppressor gene or genes on this chromosome, although not as predisposing genes in families