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Summary The initiation of a cultured human colon carcinoma line on a feeder layer of confluent fibroblasts is described. Attempts to initiate cultures without fibroblast feeder layers were not successful. Two sub-lines (designated HCT C and HCT C Col) were isolated and weaned from cells growing on the surface of the feeder layer. The sub-lines had different morphologies, secreted different levels of carcinoembryonic antigen (CEA) into the medium of confluent cultures and had slightly different karyotypes. Both sub-lines grew in semi-solid medium and formed xenografts when injected s.c. in to athymic nude mice. Analysis of radioiodinated cell membrane components indicated small, but significant differences between the sub-lines.Feeder layers of normal cells have frequently been utilized as an aid in the establishment of tissue cultures from a variety of human tumour specimens including mammary carcinoma (Smith et al., 1981), lymphoma (Epstein and Kaplan, 1979) and retinoblastoma (Gallie et al., 1982). Recently, we described the establishment of human colonic carcinomas in tissue culture (Brattain et al., 1981a). We observed that some primary cultures contained several types of malignant cells as judged by the different morphologies of cells growing on the surface of the fibroblast feeder layer. Heterogeneity of the malignant cell compartment comprising human solid tumours is currently recognized as a potentially important obstacle in several therapeutic treatment modalities of cancer Heppner et al., 1978). In addition to the clinical problems posed by heterogeneity of the malignant cells, minority subpopulations may contribute significantly to the determination of such biological properties of the tumour as metastatic or invasive abilities (Fidler, 1978;Poste & Fidler, 1980). Consequently, the identification and characterization of subpopulations of malignant cells from individual tumours is of potential importance to both the treatment and understanding of cancer. Utilizing a feeder layer of normal human colon fibroblasts we have identified two morphological subpopulations of cells from primary tissue cultures of a human colon tumour. We describe the establishment, isolation and characterization of two sub-lines of malignant cells from these primary cultures. Materials and methodsTissue culture A human colon carcinoma (designated HCT C) and normal human colon were provided by the UAB Tissue Procurement Service. The sample of normal colon was obtained at the time of colon resection for carcinoma in February, 1979 while the resection for HCT C was performed in April, 1979 on a different patient. Procedures for preparing the specimens for culture were identical and have been described for colon carcinomas in detail (Brattain et al., 1977a, b;1979;Brattain, 1979). Briefly, the specimens were minced to small pieces (1-2 mm3) and extensively washed in McCoy's tissue culture medium supplement with 20% fetal bovine serum (FBS) and antibiotics (4.3 /ugml-P gentamicin, 90 jugml-l streptomycin, 90 Uml-penicillin and 2.5...

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